Glucose/galactose biosensors and methods of using same

ABSTRACT

Provided herein are glucose and galactose biosensors and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/555,064, filed Aug. 31, 2017, which is a national stage applicationfiled under 35 U.S.C. § 371, of International Patent Application No.PCT/US2016/021073, filed Mar. 4, 2016, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 62/128,592, filedMar. 5, 2015, the contents of each of which are incorporated herein byreference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The entire content of the text file named “035327-520N01 US UPDATEDSequence Listing.txt”, which was created on Mar. 2, 2020 and is 225 KBin size, is hereby incorporated herein by reference for all purposes.

FIELD

This disclosure relates to compositions and methods for detecting anddetermining the concentration of glucose and/or galactose.

INTRODUCTION

Glucose monitoring is essential for the management of diabetes mellitus,a disease that affects at least 366 million people worldwide.Reagentless fluorescent sensors based on engineered bacterialperiplasmic glucose-binding proteins into which a single,environmentally sensitive fluorophore has been introduced are emergingas promising next-generation glucose sensors. Unlike traditionalenzyme-based glucose sensors, one key advantage of reagentless sensorsis that their monitoring mechanism requires neither additionalsubstrates for a signal to develop, nor measurement of substrateconsumption or product generation rates to deduce glucoseconcentrations. Instead, the signal originates from the response of theattached fluorophore to a glucose-induced conformational change in theprotein, which is in dynamic equilibrium with the environment withinwhich the sensor has been placed. This mechanism therefore reportsnear-instantaneously on the sample glucose concentration and itsfluctuations. Pathophysiological blood glucose concentrations range from˜3-30 mM, with euglycemia at ˜6 mM and the hyperglycemic-hyperosmoticrange at greater than about 30 mM. Improved glucose sensors and methodsfor monitoring glucose at physiological levels are desirable.

SUMMARY

Provided herein are biosensors. The biosensor may include a) apolypeptide comprising a ligand binding site and (i) one or moremutations as compared to SEQ ID NO:112 (wild-type E. coli GGBP) thatalter the ligand binding affinity of the polypeptide; and b) a reporterconjugated to the polypeptide, wherein when the polypeptide consists ofa single mutation, the single mutation is F16C, wherein the ligand-boundbiosensor results in a reporter-generated signal that is different fromthe unbound biosensor, and wherein the ligand is selected from the groupconsisting of glucose, galactose, and a combination thereof.

In some embodiments, the reporter is conjugated to F16C. In someembodiments, the polypeptide further comprises (ii) at least oneadditional mutation that replaces an amino acid with a cysteine. In someembodiments, the reporter is conjugated to the cysteine. In someembodiments, the biosensor comprises a single reporter. In someembodiments, the reporter comprises a fluorophore and wherein the signalis a fluorescent signal. In some embodiments, the fluorophore isselected from the group consisting of acrylodan and badan. In someembodiments, the signal comprises an emission intensity of thefluorophore recorded at one or more wavelengths. In some embodiments,the change in signal comprises a shift in the one or more wavelengths.In some embodiments, the signal comprises a ratio of emissionintensities recorded at two or more wavelengths. In some embodiments,the change in signal comprises a shift in two or more wavelengths. Insome embodiments, the at least one additional mutation (ii) is W183C. Insome embodiments, the reporter is conjugated to W183C. In someembodiments, each mutation (i) is a mutation to an amino acid selectedfrom the group consisting of Y10, D14, F16, N91, K92, E149, H152, D154,A155, R158, M182, N211, D236, and N256, and combinations thereof. Insome embodiments, each mutation (i) is selected from the groupconsisting of Y10A, D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L,D14Y, D14F, F16L, F16A, N91A, K92A, E149K, E149Q, E149S, H152A, H152F,H152Q, H152N, D154A, D154N, A155S, A155H, A155L, A155F, A155Y, A155N,A155K, A155M, A155W, A155Q, R158A, R158K, M182W, N211F, N211W, N211K,N211Q, N211S, N211H, N211M, D236A, D236N, N256A, and N256D, andcombinations thereof. In some embodiments, the mutation (i) affects theinteraction of the polypeptide with bound glucose, wherein theinteraction is with a portion of the glucose selected from the groupconsisting of 1-hydroxyl, 2-hydroxyl, 3-hydroxyl, 4-hydoxyl, 6-hydroxyl,pyranose ring, and combinations thereof. In some embodiments, themutation (i) affects the interaction of the mutant polypeptide with thereporter group. In some embodiments, the mutation (i) affects theinteraction of the mutant polypeptide with a water molecule. In someembodiments, the polypeptide has an affinity (KD) for glucose within theconcentration range of glucose in vivo for a subject. In someembodiments, the polypeptide has an affinity (KD) for galactose withinthe concentration range of galactose in vivo for a subject. In someembodiments, the subject is a mammal. In some embodiments, the subjectis a primate or non-primate. In some embodiments, the subject is anon-primate selected from a cow, pig, camel, llama, horse, goat, rabbit,sheep, hamsters, guinea pig, cat, dog, rat, and mouse. In someembodiments, the subject is a primate selected from a monkey,chimpanzee, and human. In some embodiments, the subject is a human.

In some embodiments, the polypeptide has an affinity (KD) for glucose inthe range of about 0.2 mM to about 100 mM. In some embodiments, thepolypeptide has an affinity (KD) for galactose in the range of about 0.8mM to about 100 mM. In some embodiments, the biosensor is capable ofdetecting glucose in the hypoglycemic, hyperglycemic, andhyperglycemic-hyperosmotic ranges. In some embodiments, the biosensor iscapable of detecting glucose in the range of about 0.1 mmol/L to about120 mmol/L. In some embodiments, the biosensor is capable of detectingglucose in the range of about 4 mmol/L to about 33 mmol/L. In someembodiments, the biosensor is capable of detecting galactose in therange of about 0.2 mM to about 400 mM.

In some embodiments, the mutant polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-54.

Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 1. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:3. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 4. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 5. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:6. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 7. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 8. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:9. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 10. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 11. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:12. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 13. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 14. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:15. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 16. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 17. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:18. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 19. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 20. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:21. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 22. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 23. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:24. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 25. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 26. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:27. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 28. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 29. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:30. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 31. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 32. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:33. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 34. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 35. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:36. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 37. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 38. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:39. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 40. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 41. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:42. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 43. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 44. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:45. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 46. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 47. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:48. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 49. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 50. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:51. Further provided herein is a polypeptide comprising the amino acidsequence of SEQ ID NO: 52. Further provided herein is a polypeptidecomprising the amino acid sequence of SEQ ID NO: 53. Further providedherein is a polypeptide comprising the amino acid sequence of SEQ ID NO:54. Further provided herein is a polynucleotide encoding the polypeptideas described herein. In some embodiments, the polynucleotide comprisesat least one sequence selected from the group consisting of SEQ ID NOs:56-109.

Further provided herein is a vector comprising the polynucleotide ofclaim 88 or 89.

Further provided herein is a panel comprising a plurality of biosensorsas described herein. In some embodiments, the panel comprises acomposite sensor or an array. In some embodiments, the array is selectedfrom a multichannel array or multiplexed array. In some embodiments,each biosensor comprises the same reporter group. In some embodiments,each biosensor comprises a different reporter group. In someembodiments, the array comprises a plurality of sensor elements, eachsensor element comprising a biosensor different from or the same asthose of the other sensor elements. In some embodiments, the compositesensor comprises a plurality of sensor elements, each sensor elementcomprising a mixture of different biosensors. In some embodiments, thecomposite sensor comprises a single sensor element, the single sensorelement comprising a mixture of different biosensors.

Further provided herein is a method of determining the concentration ofglucose, galactose, or a combination thereof, in a sample from asubject. The method may include applying the sample to a panelcomprising a plurality of biosensors as described herein. In someembodiments, the sample is from a subject. In some embodiments, thesample comprises a biological fluid. In some embodiments, the biologicalfluid is selected from the group consisting of blood, urine,interstitial fluid, saliva, sweat, tears, gastric lavage, fecal matter,emesis, bile, or combinations thereof. In some embodiments, the samplecomprises skin.

Further provided herein is a method of detecting the presence of aligand in a sample. The method may include a) contacting the biosensoras described herein with the sample; b) measuring a signal from thebiosensor; and c) comparing the signal to a ligand-free control, whereina difference in signal indicates the presence of ligand in the sample,and wherein the ligand is selected from the group consisting of glucose,galactose, and a combination thereof.

Further provided herein is a method of determining the concentration ofa ligand in a sample. The method may include a) contacting the biosensoras described herein with the sample; b) measuring a signal from thebiosensor; and c) comparing the signal to a standard hyperbolic ligandbinding curve to determine the concentration of ligand in the testsample, wherein the standard hyperbolic ligand binding curve is preparedby measuring the signal transduced by the biosensor when contacted withcontrol samples containing known concentrations of ligand, and whereinthe ligand is selected from the group consisting of glucose, galactose,and a combination thereof.

Further provided herein is a method of episodically or continuouslymonitoring the presence of a ligand in a reaction. The method mayinclude a) contacting the biosensor as described herein with thereaction; b) maintaining the reaction under conditions such that thepolypeptide is capable of binding ligand present in the reaction; and c)episodically or continuously monitoring the signal from the biosensor inthe reaction, wherein the ligand is selected from the group consistingof glucose, galactose, and a combination thereof.

Further provided herein is a method of episodically or continuouslymonitoring the presence of a ligand in a reaction. The method mayinclude a) contacting the biosensor as described herein with thereaction; b) maintaining the reaction under conditions such that thepolypeptide is capable of binding ligand present in the reaction; c)episodically or continuously monitoring the signal from the biosensor inthe reaction; and d) comparing the signal to a standard hyperbolicligand binding curve to determine the concentration of ligand in thetest sample, wherein the standard hyperbolic ligand binding curve isprepared by measuring the signal transduced by the biosensor whencontacted with control samples containing known concentrations ofligand, wherein the ligand is selected from the group consisting ofglucose, galactose, and a combination thereof.

In some embodiments, the biosensor is placed in contact with a subject'sskin or mucosal surface. In some embodiments, the biosensor is implantedin a subject's body. In some embodiments, the biosensor is implanted ina subject's blood vessel, vein, eye, natural or artificial pancreas,alimentary canal, stomach, intestine, esophagus, or skin. In someembodiments, the biosensor is configured within or on the surface of acontact lens. In some embodiments, the biosensor is configured to beimplanted in the skin. In some embodiments, the biosensor is implantedin a subject with an optode. In some embodiments, the biosensor isimplanted in a subject with a microbead. In some embodiments, thebiosensor generates the signal transdermally. In some embodiments, themethod further includes d) comparing the signal to a ligand-freecontrol, wherein a difference in signal indicates the presence of ligandin the reaction. In some embodiments, the method further includes d)comparing the signal to a standard hyperbolic ligand binding curve todetermine the concentration of ligand in the test sample, wherein thestandard hyperbolic ligand binding curve is prepared by measuring thesignal transduced by the biosensor when contacted with control samplescontaining known concentrations of ligand. In some embodiments, thesample comprises a fermentation sample. In some embodiments, the samplecomprises food or beverage. In some embodiments, the sample comprises abeverage selected from soft drink, fountain beverage, water, coffee,tea, milk, dairy-based beverage, soy-based beverage, almond-basedbeverage, vegetable juice, fruit juice, fruit juice flavored drink,energy drink, sport drink, and alcoholic product, and combinationsthereof. In some embodiments, the sample comprises water selected fromflavored water, mineral water, spring water, sparkling water, and tonicwater, and combinations thereof. In some embodiments, the samplecomprises an alcoholic product selected from beer, malt beverage,liqueur, whiskey, and wine, and combinations thereof. In someembodiments, the sample comprises food comprising a semi-solid or liquidform. In some embodiments, the sample comprises food selected fromyogurt, soup, ice cream, broth, purees, shakes, smoothies, batter,condiments, and sauce, and combinations thereof. In some embodiments,the sample is from food engineering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the overlay of the wild-type GGBP(turquoise) glucose complex with acrylodan (orange) conjugated to theW183C mutant (green). Calcium (magenta) binds to one of the two proteindomains.

FIG. 2 is a drawing showing the interaction between the protein andbound glucose in the 183C-acrylodan conjugate.

FIG. 3 is a layout of a Tecan Genesis liquid-handling robot for settingup complex high-precision titration series in accordance with oneembodiment of the present disclosure.

FIG. 4 is a schematic of a liquid-handling software environmentaccording to one embodiment of the present disclosure. Multi-componenttitrations involve the addition of several constant components, andlogarithmic titration series of one or multiple titrants. The titrationsare set up in scripts or graphical user interfaces, and translated intomachine instructions in a two-stage manner.

FIGS. 5A˜5F are graphs showing different landscapes of 183C-acrylodan[(A)-(C)] and BD_SM4⋅acrylodan [(D)-(F)] fluorescence signals (R, ratioof emission intensities at 440 nm and 510 nm) as a function of glucoseconcentration and temperature (the data comprises 3840 observations: 48glucose concentrations, 80 temperatures). The changes in fluorescencearise as the system exchanges between three major states: A, foldedapoprotein; S, folded saturated glucose complex; D, denaturedapo-protein. The denatured state undergoes a furthertemperature-dependent two-state conformational change, splitting intosub-states D1 and D2. In 183C-Acrylodan the saturated glucose complexundergoes a temperature-dependent conformation change, splitting into S1and S2 sub-states. Dotted lines indicate approximate mid-points ofunfolding, ligand binding, and conformational changes.

FIG. 6 is a schematic representation of the binding, confirmationchange, and unfolding equilibria in GGBP and their sensitivity toglucose binding at constant calcium. The leaves of the tree correspondto experimentally observable states of the protein; the nodes are theequilibria between these individual states, or ensembles composed ofthese states. All equilibria are temperature sensitive. Bindingequilibria put energy into the system, which propagates upward into thevarious ensembles (red arrows). Symbols are defined in thespecification.

FIGS. 7A˜7B are graphs showing the estimation of apo-protein stability.The experimental dependence of the fluorescence signal, R, is fit to atwo-state model over the temperature range where the thermaldenaturation between A and D1 dominates. (A) GBP183C Acrylodan (RMSD;apoTm, 322.2 K; Δapo Hu, 197.3 kcal/mol). (B) BD_SM4 Acrylodan (apoTm,329.7 K; ΔapoHu, 156.6 kcal/mol).

FIG. 8 are graphs showing glucose binding isotherm at 37° C. (310K).Left column: GBP183C•Acrylodan; right column: BD_SM4•Acrylodan. Greenline, fit of equation 18 to the data: Kd values of 6.58±0.03 mM and4.89±0.02 mM for GBP183C•Acrylodan and BD_SM4•Acrylodan respectively.The RMSD values of 0.00418 and 0.0044 correspond to estimated errors of0.48% (GBP183C•Acrylodan) and 0.48% (BD_SM4•Acrylodan) at[glucose]=Kd•Top: the signal with respect to the logarithm of glucoseconcentration shows even sampling of the binding isotherm (the zeroligand point is placed at 10-7 M for visualization). Bottom: the lineardependence of the signal on glucose concentration emphasizes the effectof high glucose concentrations on the fluorescence baseline.

FIG. 9 are graphs showing the temperature dependence of glucose binding.Left column, GBP183C•Acrylodan; right column, BD_SM4•Acrylodan. Top row:temperature dependence of glucose binding free energy (green, fit toGibbs-Helmholtz equation 8). Bottom row: temperature dependence of theglucose dissociation constant (green, transform of the fitGibbs-Helmholtz curve).

FIGS. 10A˜10B are graphs showing the conformational changes in thefolded protein-glucose complex of GBP183C•acrylodan. (A) Thermal meltsare shown for the subset of individual wells containing 0.2-1.8 Mglucose, corresponding to saturated protein-ligand complexes. Thesaturated complex undergoes a thermal conformational change, separatingits baseplane into two components, S1 and S2. (B) The glucose osmolyteeffect on this conformation. Tc values calculated by individual fits (+)fit to a linear dependence on glucose concentrations (blue line). (C)ΔHc values calculated by individual fits (+) fit to a linear dependenceon glucose concentrations (blue line).

FIGS. 11A˜11D are graphs showing global fits of the thermodynamic modelto the fluorescence ratio (R) dependence on glucose and temperature at 1mM CaCl₂) for GBP183C•Acrylodan (A) and BD_SM4•Acrylodan (C): blue,observations; red, model; contours, residuals. Bootstrap analysis (B andD) shows the average model (orange), and range of deviation in thevalues (contours).

FIG. 12 are graphs showing the fluorescence response ofGGBP183C•acrylodan (top) and BD_SM4•acrylodan (bottom) to Ca2+. Althoughthere is no direct response to binding Ca2+, the stability of proteinshifts in response.

FIG. 13 are graphs showing the thermal stability shifts upon addition ofCa2+ to GGBP183C•acrylodan (top) and BD_SM4•acrylodan (bottom) in theabsence of glucose. Green crosses, Tm (left) and ΔHu (right) valuesextracted from the experimental data; blue lines, calculated values. Thecalculated Kd values at 37° C. for GGBP183C•acrylodan are: single foldedstate site, 10.8 nM; single, medium-affinity denatured state, 0.37 mM;two, low-affinity denatured states 8.8 mM. The corresponding values forBD_SM4•acrylodan are 12.2 nM, 4.3 mM, and 11.9 mM.

FIG. 14 are graphs showing the parameters extracted from landscapes thatrecord the dependence of fluorescence on glucose concentrations andtemperature at a given calcium concentration for GGBP183C•acrylodan(red) and BD_SM4•acrylodan (blue). Lines are drawn as a guide to trendsonly.

FIG. 15 are graphs showing the dependence of Ca2+ affinity on glucosefor GGBP183C•acrylodan (left) and BD_SM4•acrylodan (right). Extractionof Tm and ΔHu values from the GGBP183C•acrylodan at non-zero glucoseconcentrations is confounded by effects on baselines by theconformational change in the glucose-saturated complex.

FIG. 16 is a schematic showing the representation of the binding,conformational change, and unfolding equilibria in GGBP and theirsensitivity to glucose and calcium binding (also see FIG. 6 ).

FIG. 17 is a graph showing the systematic calibration error due to thetemperature dependence of glucose binding. Depicted is the variation ofglucose concentration corresponding to a constant signal (isochrome) asa function of temperature. Tcal is the calibration temperature. Tobs,the actual temperature; Lcal and Ltrue the associated glucoseconcentrations.

FIG. 18 is a graph showing the systematic temperature-dependent error inglucose concentration, ε(T), was calculated for BD_SM4•acrylodan in the4-10 mM interval. The projection of this surface is shown, indicatingthat dε(T)/dT ˜3.7% K−1.

FIG. 19 is a drawing showing mutations at positions 149 (unfavorableinteraction with acrylodan), 152 (hydrogen bond to glucose 6-OH), and155 (interaction with water) tune the glucose affinity.

FIGS. 20A˜20B3 are drawings showing the ratioametric fluorescent signalresponses with respect to temperature and glucose of the 183C-acrylodanconjugates of seven mutants that respond in the pathophysiologicalconcentration range (see Tables 1˜4 for details of their ligand-bindingaffinities). For each mutant, two different views are shown: athree-dimensional perspective to illustrate the qualitative character ofthe fluorescent landscape, and a projection to enable direct comparisonbetween the landscapes.

FIG. 21 is a schematic that illustrates over what glucose concentrationranges each sensor is expected to perform with high accuracy.

FIG. 22 is a drawing showing the ratiometric signal response withrespect to temperature and glucose concentration of the 16C-acrylodanand 16C-badan conjugates.

FIG. 23 is a schematic map of the polynucleotide sequence (SEQ ID NO:113 and SEQ ID NO: 114) of an expression vector comprising thepolynucleotide sequence encoding the polypeptide sequence of wild-typeE. coli GGBP (SEQ ID NO: 55) and including poly-histidine polypeptide ofsequence GGSHHHHHH (SEQ ID NO: 111).

DETAILED DESCRIPTION

Described herein are novel engineered biosensors. These biosensors mayhave altered ligand-binding affinities, tailored ligand-bindingspecificities, and/or temperature dependencies of ligand binding orstability. For example, the herein described engineered glucose andgalactose biosensors provide high-accuracy information related toextended glucose concentration ranges. The glucose concentration rangesexpand over three orders of magnitude and may include thepathophysiological hypo- and hyper-glycemic range in humans. Methods ofmaking these glucose/galactose biosensors, and methods of using theseglucose/galactose biosensors are also described herein.

1. Definitions

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The term “about” as used herein as applied to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain aspects, the term “about” refers to a range of values thatfall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

“Amino acid” as used herein refers to naturally occurring andnon-natural synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code. Amino acids can be referred to herein by eithertheir commonly known three-letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission. Aminoacids include the side chain and polypeptide backbone portions.

The terms “control,” “reference level,” and “reference” are used hereininterchangeably. The reference level may be a predetermined value orrange, which is employed as a benchmark against which to assess themeasured result. The predetermined level may be from a subject or agroup or a composition of known ligand concentration. “Control group” asused herein refers to a group of control subjects. The predeterminedlevel may be a cutoff value from a control group. The predeterminedlevel may be an average from a control group. Cutoff values (orpredetermined cutoff values) may be determined by Adaptive Index Model(AIM) methodology. Cutoff values (or predetermined cutoff values) may bedetermined by a receiver operating curve (ROC) analysis from biologicalsamples of the patient group. ROC analysis, as generally known in thebiological arts, is a determination of the ability of a test todiscriminate one condition from another, e.g., to determine theperformance of each marker in identifying a patient having CRC. Adescription of ROC analysis is provided in P. J. Heagerty et al.(Time-dependent ROC curves for censored survival data and a diagnosticmarker, Biometrics 2000, 56, 337-44), the disclosure of which is herebyincorporated by reference in its entirety. Alternatively, cutoff valuesmay be determined by a quartile analysis of biological samples of apatient group. For example, a cutoff value may be determined byselecting a value that corresponds to any value in the 25th-75thpercentile range, preferably a value that corresponds to the 25thpercentile, the 50th percentile or the 75th percentile, and morepreferably the 75th percentile. Such statistical analyses may beperformed using any method known in the art and can be implementedthrough any number of commercially available software packages (e.g.,from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station,Tex.; SAS Institute Inc., Cary, N.C.). The healthy or normal levels orranges for a ligand may be defined in accordance with standard practice.

The term “expression vector” indicates a plasmid, a virus or anothermedium, known in the art, into which a nucleic acid sequence forencoding a desired protein can be inserted or introduced.

“Food engineering” refers to the multidisciplinary field of appliedphysical sciences which combines science, microbiology, and engineeringfor food and related industries. Food engineering includes, but is notlimited to, the application of agricultural engineering, mechanicalengineering, and chemical engineering principles to food materials toallow for food processing, food machinery, packaging, ingredientmanufacturing, instrumentation, and control. For example, foodengineering may include the natural sweetening of food products byengineering Lactococcus lactis for glucose production, whereby theglucose/galactose biosensors provided herein may be used tointermittently and/or continuously monitor the level of glucoseproduction (see, for example, Pool et al. Metabolic Engineering 2006, 8,456-464).

The term “host cell” is a cell that is susceptible to transformation,transfection, transduction, conjugation, and the like with a nucleicacid construct or expression vector. Host cells can be derived fromplants, bacteria, yeast, fungi, insects, animals, etc.

“Ligand” refers to the entity whose presence and/or concentration may bedetermined using the biosensor. Accordingly, a ligand may also bereferred to as a substrate or analyte. As used herein, the ligandcomprises glucose, galactose, or a combination thereof. In someembodiments, the ligand comprises glucose. In some embodiments, theligand comprises galactose. In some embodiments, the ligand may be amodified glucose or galactose, such as, for example, glucose orgalactose including a label such as a radioactive or fluorescent label.

“Polynucleotide” as used herein can be single stranded or doublestranded, or can contain portions of both double stranded and singlestranded sequence. The polynucleotide can be nucleic acid, natural orsynthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where thepolynucleotide can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,and isoguanine. Polynucleotides can be obtained by chemical synthesismethods or by recombinant methods.

A “peptide” or “polypeptide” is a linked sequence of amino acids and canbe natural, synthetic, or a modification or combination of natural andsynthetic. Peptides and polypeptides include proteins such as bindingproteins. “Primary structure” refers to the amino acid sequence of aparticular peptide. “Secondary structure” refers to locally ordered,three dimensional structures within a polypeptide. These structures arecommonly known as domains, e.g., enzymatic domains, extracellulardomains, transmembrane domains, pore domains, and cytoplasmic taildomains. Domains are portions of a polypeptide that form a compact unitof the polypeptide and are typically 15 to 350 amino acids long.Exemplary domains include domains with enzymatic activity or ligandbinding activity. Typical domains are made up of sections of lesserorganization such as stretches of beta-sheet and alpha-helices.“Tertiary structure” refers to the complete three dimensional structureof a polypeptide monomer. “Quaternary structure” refers to the threedimensional structure formed by the noncovalent association ofindependent tertiary units.

“Sample” or “test sample” as used herein can mean any sample in whichthe presence and/or level of glucose and/or galactose is to be detectedor determined. Samples may include liquids, solutions, emulsions, orsuspensions. Samples may include a reaction for episodic or continuousmonitoring. Samples may include industrial samples such as from foodindustry or food engineering. Samples may include fermentation samples,food, or beverages. Food and beverage may include any edible and/orpotable liquid, solution, emulsion, or suspension. Food may comprise asemi-solid or liquid form. Food may include, for example, yogurt, soup,ice cream, broth, purees, shakes, smoothies, batter, condiments, sauce,and combinations thereof. Beverages may include, for example, softdrinks, fountain beverages, waters, coffee, tea, milk, dairy-basedbeverages, soy-based beverages, almond-based beverages, vegetable juice,fruit juice, fruit juice flavored drinks, energy drinks, sport drinks,or alcoholic products, and combinations thereof. Waters may include, forexample, flavored water, mineral water, spring water, sparkling water,and tonic water, and combinations thereof. Alcoholic products mayinclude, for example, beer, malt beverages, liqueurs, whiskeys, or wine,and combinations thereof. In some embodiments, the sample includes aglucose-containing beverage. Samples may include a medical sample.Samples may include any biological fluid or tissue, such as blood, wholeblood, fractions of blood such as plasma and serum, muscle, interstitialfluid, sweat, saliva, urine, tears, synovial fluid, bone marrow,cerebrospinal fluid, nasal secretions, sputum, amniotic fluid,bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lungtissue, peripheral blood mononuclear cells, total white blood cells,lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells,bile, digestive fluid, skin, or combinations thereof. In someembodiments, the sample comprises an aliquot. In some embodiments, thesample comprises skin (e.g., transdermal glucose monitoring). In otherembodiments, the sample comprises a biological fluid. Samples can beobtained by any means known in the art.

By “specifically binds,” it is generally meant that a polypeptide orbiosensor, or derivative thereof, binds to a cognate ligand or analytein preference to a random, unrelated ligand or interferent.

“Subject” as used herein refers to any subject, particularly a mammaliansubject, who wants to or is in need of detecting ligand or determiningthe concentration of ligand with the herein described biosensors. Thesubject may be a human or a non-human animal. The subject may be amammal. The mammal may be a primate or a non-primate. The mammal can bea primate such as a human; a non-primate such as, for example, dog, cat,horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster,and guinea pig; or non-human primate such as, for example, monkey,chimpanzee, gorilla, orangutan, and gibbon. The subject may be of anyage or stage of development, such as, for example, an adult, anadolescent, or an infant.

“Substantially identical” can mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100 amino acids.

“Variant” used herein with respect to a polynucleotide means (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a polynucleotide that is substantially identical to a referencedpolynucleotide or the complement thereof; or (iv) a polynucleotide thathybridizes under stringent conditions to the referenced polynucleotide,complement thereof, or a sequences substantially identical thereto.

A “variant” can further be defined as a peptide or polypeptide thatdiffers in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. Representative examples of “biological activity”include the ability to be bound by a specific antibody or to promote animmune response. Variant can mean a substantially identical sequence.Variant can mean a functional fragment thereof. Variant can also meanmultiple copies of a polypeptide. The multiple copies can be in tandemor separated by a linker. Variant can also mean a polypeptide with anamino acid sequence that is substantially identical to a referencedpolypeptide with an amino acid sequence that retains at least onebiological activity. A conservative substitution of an amino acid, i.e.,replacing an amino acid with a different amino acid of similarproperties (e.g., hydrophilicity, degree and distribution of chargedregions) is recognized in the art as typically involving a minor change.These minor changes can be identified, in part, by considering thehydropathic index of amino acids. See Kyte et al., J. Mol. Biol. 1982,157, 105-132. The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophibicity of aminoacids can also be used to reveal substitutions that would result inpolypeptides retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a polypeptide permitscalculation of the greatest local average hydrophilicity of thatpolypeptide, a useful measure that has been reported to correlate wellwith antigenicity and immunogenicity, as discussed in U.S. Pat. No.4,554,101, which is fully incorporated herein by reference. Substitutionof amino acids having similar hydrophilicity values can result inpolypeptides retaining biological activity, for example immunogenicity,as is understood in the art. Substitutions can be performed with aminoacids having hydrophilicity values within ±2 of each other. Both thehydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant can be a polynucleotide sequence that is substantiallyidentical over the full length of the full gene sequence or a fragmentthereof. The polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical over the full length of the gene sequence or afragment thereof. A variant can be an amino acid sequence that issubstantially identical over the full length of the amino acid sequenceor fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical over the full length of the amino acidsequence or a fragment thereof.

2. Biosensor

Biosensors are molecular recognition elements that transduceligand-binding events into physical signals. Biosensors as detailedherein bind at least one ligand and emit a signal. A ligand-boundbiosensor results in a signal that is different from the unboundbiosensor. This difference facilitates detection of the at least oneligand and/or determination of ligand concentration. The biosensors maybe used without the assistance of other reagents. The measuring ordetecting process does not change the composition of the sensor, unlikeenzyme-based or competitive displacement assays. The biosensor comprisesa polypeptide and at least one reporter group. The polypeptide may haveone or more mutations as compared to a corresponding wild-typepolypeptide.

a. Polypeptide

According to the compositions and methods detailed herein, thepolypeptide comprises a mutant glucose-galactose binding protein (GGBP)from Escherichia coli. GGBP is a member of the bacterial periplasmicbinding protein (PBP) superfamily that mediates chemotaxis and uptake ofa wide variety of chemical species, including sugars, amino acids,oligopeptides, and metals. PBPs include two domains connected by a hingeregion, with a ligand binding site located at the interface between thetwo domains. The ligand binding site can adopt two differentconformations: a ligand-free open form, and a ligand-bound closed form.The ligand-free open form and ligand-bound closed form interconvertthrough a bending motion around the hinge.

In some embodiments, the polypeptide comprises at least one mutationcompared to a wild-type glucose-galactose binding protein fromEscherichia coli. In some embodiments, the wild-type glucose-galactosebinding protein from Escherichia coli is encoded by a polynucleotide ofSEQ ID NO: 110 and comprises an amino acid sequence of SEQ ID NO: 55. Insome embodiments, the wild-type glucose-galactose binding protein fromEscherichia coli comprises an amino acid sequence of SEQ ID NO: 112. SEQID NO: 112 corresponds to SEQ ID NO: 55 without the starting methionine.

The polypeptide includes at least one binding site for at least oneligand. The polypeptide binds the at least one ligand. In someembodiments, the polypeptide binds a single ligand. In some embodiments,the polypeptide binds two ligands.

In some embodiments, the polypeptide may include a purification tag. Thepurification tag may comprise, for example, poly-histidine,glutathione-S-transferase (GST), poly-glutamate, calmodulin, FLAG,Xpress, and combinations thereof. The purification tag may be present atthe C-terminus of the polypeptide, the N-terminus of the polypeptide, aninternal location in the amino acid sequence of the polypeptide, or acombination thereof. In some embodiments, the polypeptide comprises apurification tag comprising a poly-histidine polypeptide of sequenceGGSHHHHHH (SEQ ID NO: 111).

i. Mutations

The polypeptide comprises at least one mutation as compared to wild-typeE. coli GGBP (SEQ ID NO: 112). SEQ ID NO: 112 corresponds to SEQ ID NO:55 without the starting methionine. The numbering of the mutationscorresponds to the numbering of the amino acids in SEQ ID NO: 112. Insome embodiments, the polypeptide comprises one or more mutations ascompared to wild-type E. coli GGBP (SEQ ID NO: 112). In someembodiments, the polypeptide comprises two or more mutations as comparedto wild-type E. coli GGBP (SEQ ID NO: 112).

1) Affinity-Tuning

The polypeptides as detailed herein include at least one mutation thatalters the ligand binding affinity of the polypeptide. In someembodiments, the affinity-altering mutations include changes to aminoacids in the ligand binding site. The affinity-altering mutations maymutate an amino acid that interacts with a portion of the ligand. Whenthe ligand is glucose, the interaction affected may be between the aminoacid and a portion of the glucose molecule including, for example,1-hydroxyl, 2-hydroxyl, 3-hydroxyl, 4-hydoxyl, 6-hydroxyl, pyranosering, and combinations thereof. The affinity-altering mutations maymutate an amino acid that interacts with the reporter group. Theaffinity-altering mutations may mutate an amino acid that interacts witha water molecule.

Mutations that alter the GGBP glucose affinity while enabling theextension of high accuracy coverage in the hyperglycemic to hypoglycemicconcentration ranges can be divided into three classes: (Class A)mutations that impact the direct interactions of the GGBP with theglucose hydroxyls and/or pyranose ring; (Class B) mutations that impactthe interaction of the GGBP with the fluorophore; and/or (Class C)mutations that impact the interaction of the GGBP with the buried watermolecule present in the fluorescent conjugate, but not the wild-typeprotein. These mutations can be constructed by site-directedmutagenesis, total gene synthesis or semi-synthesis, with specificresidues being mutated.

Class A mutations include those of, for example, polypeptides GGBP183C(SEQ ID NO: 1), GGBP16C (SEQ ID NO: 2), GGBP183C14A (SEQ ID NO: 3),GGBP183C152N (SEQ ID NO: 4), GGBP183C152F (SEQ ID NO: 5), andGGBP183C152Q (SEQ ID NO: 6).

Class B mutations include those of, for example, polypeptidesGGBP183C149Q (SEQ ID NO: 7), GGBP183C149S (SEQ ID NO: 8), andGGBP183C149K (SEQ ID NO: 9).

Class C mutations include those of, for example, polypeptidesGGBP183C155N (SEQ ID NO: 10) and GGBP183C155H (SEQ ID NO: 11).

The affinity-altering mutation may result in a biosensor having anaffinity (K_(D)) for ligand within the physiological range of the ligandin a subject. The physiological range of the ligand in a subject mayinclude normal or healthy levels. When the ligand comprises glucose, thephysiological range of the ligand in a subject may include normalranges, hypoglycemic ranges hyperglycemic ranges, andhyperglycemic-hyperosmotic ranges of glucose.

The normal range of glucose concentration in blood for humans may beabout 60 mg/dL to about 140 mg/dL, 100 mg/dL to about 140 mg/dL, orabout 60 mg/dL to about 90 mg/dL. The hypoglycemic range for humans maybe less than about 3.9 mM, less than about 3.3 mM, less than about 2.8mM, less than about 2.2 mM, less than about 70 mg/dL, less than about 60mg/dL, less than about 50 mg/dL, or less than about 40 mg/dL. Thehyperglycemic range for humans may be greater than about 20 mM, greaterthan about 15 mM, greater than about 11.1 mM, greater than about 7 mM,greater than about 5.6 mM, greater than about 300 mg/dL, greater thanabout 250 mg/dL, greater than about 200 mg/dL, greater than about 126mg/dL, or greater than about 100 mg/dL. The hyperosmolar-hypoglycemicrange for humans may be greater than about 600 mg/dL. In someembodiments, the biosensor is able to detect glucose in a concentrationrange of about 2-4 mM (hypoglycemic) to about 10-33 mM (hyperglycemic)to greater than about 33 mM (hyperosmolar-hypoglycemic). Theconcentration of glucose in the blood may also be referred to as bloodsugar level.

In some embodiments, the biosensor has an affinity (K_(D)) for glucosewithin the physiological range of the ligand in a subject. In someembodiments, the biosensor has an affinity (K_(D)) for glucose of about0.2 mM to about 100 mM. In some embodiments, the biosensor is capable ofdetecting glucose in the range of about 0.1 mmol/L to about 120 mmol/L.In some embodiments, the biosensor is capable of detecting glucose inthe range of about 4 mmol/L to about 33 mmol/L.

In some embodiments, the biosensor has an affinity (K_(D)) for galactosewithin the physiological range of the ligand in a subject. In someembodiments, the biosensor has an affinity (K_(D)) for galactose ofabout 0.8 mM to about 100 mM, or about 1 mM to about 90 mM. In someembodiments, the biosensor is capable of detecting galactose in therange of about 0.2 mmol/L to about 400 mmol/L or about 0.5 mmol/L toabout 300 mmol/L.

In some embodiments, a single biosensor has an affinity (K_(D)) forglucose and/or galactose within the physiological range of the ligand ina subject. In some embodiments, a plurality of biosensors together hasan affinity (K_(D)) for glucose and/or galactose within thephysiological range of the ligand in a subject, wherein each biosensorhas an affinity (K_(D)) for glucose and/or galactose within a portion ofthe physiological range of the ligand in a subject.

In some embodiments, the polypeptide includes a mutation to an aminoacid selected from the group consisting of Y10, D14, F16, N91, K92,E149, H152, D154, A155, R158, M182, N211, D236, and N256, andcombinations thereof. In some embodiments, the polypeptide includes amutation selected from Y10A, D14A, D14Q, D14N, D14S, D14T, D14E, D14H,D14L, D14Y, D14F, F16L, F16A, N91A, K92A, E149K, E149Q, E149S, H152A,H152F, H152Q, H152N, D154A, D154N, A155S, A155H, A155L, A155F, A155Y,A155N, A155K, A155M, A155W, A155Q, R158A, R148K, M182W, N211F, N211W,N211K, N211Q, N211S, N211H, N211M, D236A, D236N, N256A, and N256D, andcombinations thereof. In some embodiments, the polypeptide comprises anamino acid sequence of any one of SEQ ID NOs: 1-54. In some embodiments,the polypeptide is encoded by a polynucleotide sequence of any one ofSEQ ID NOs: 56-109.

Affinity of the polypeptide for ligand may be determined by any meansknown by one of skill in the art. Affinity may be defined by K_(D).Affinity may be determined as exemplified in Example 3. In someembodiments, biosensor function may be assessed using fluorescenceemission biosensors recorded in the absence and presence of saturatingligand concentrations. Spectral changes may be characterized by fourparameters: wavelength shift (the difference between the wavelengths ofemission maximum in the unbound and ligand-saturated states), directionof intensity change (increase or decrease in intensity at thewavelengths of maximum emission in the two states), standard intensitychange (ΔI_(std)), and standard ratiometric change (ΔR). ΔI_(std) isdefined as the normalized intensity change relative to the averageintensity, determined at the wavelength mid-point between the twoemission maxima:

$\begin{matrix}{{\Delta I_{std}} = {❘\frac{2\left( {{I_{1}\left( \lambda_{std} \right)} - {I_{2}\left( \lambda_{std} \right)}} \right)}{{I_{1}\left( \lambda_{std} \right)} + {I_{2}\left( \lambda_{std} \right)}}❘}} & 35\end{matrix}$

where ΔI_(std)=(λ_(max, unbound)+λ_(max, saturated))/2 and I₁, I₂ arethe fluorescence intensities at λ_(std) of each spectrum respectively.ΔR is defined in terms of two emission bands, A₁([λ₁, λ₂]) and A₂([λ₃,λ₄]):

$\begin{matrix}{{\Delta R} = {❘{\frac{0A_{1}}{0A_{2}} - \frac{\infty A_{1}}{\infty A_{2}}}❘}} & 36\end{matrix}$

where ⁰A₁, ^(∞)A₂ are the areas in the absence of ligand, and ⁰A₁,^(∞)A₂ the areas in the presence of saturating ligand. A computerprogram may be used to enumerate ΔR for all possible pairs of wavelengthbands in the two spectra, to identify the optimal sensing condition,defined as the maximum value of ΔR. Adjustable parameters of thealgorithm, and their values used for ΔR_(max) quantities reported mayinclude: step size (2 nm), step width (10 nm), minimum integration arealimit (fraction of total: 0.1), and maximum integration area limit(fraction of total: 1). Affinity of a biosensor for a ligand may bedetermined by fluorimetric titration. The emission wavelength monitoredmay be that of maximum difference in intensity between the ligand-freeand bound states. For each biosensor, fluorescence intensiometricobservations may be fit to a hyperbolic binding isotherm for a two-statemodel (Marvin et al., Proc. Natl. Acad. Sci. USA 1997, 94, 4366-4371):

$\begin{matrix}{F = \frac{{K_{d}F_{F}} + {\lbrack S\rbrack F_{B}}}{K_{d} + \lbrack S\rbrack}} & 37\end{matrix}$

where F is fluorescence at ligand concentration [S], K_(d) is thedissociation constant, and F_(F), F_(B) are the fluorescence intensitiesof the ligand-free and ligand-saturated states, respectively. Forratiometric observations, equation 37 may be modified to account fordifferentially weighted contributions of the two emission bands(Lakowicz, Principles of Fluorescence Spectroscopy, 2^(nd) Ed. KluwerAcademic Press, New York, p. 698, 1999):

$\begin{matrix}{R = \frac{{appK_{d}R_{F}} + {\lbrack S\rbrack R_{B}}}{{appK_{d}} + \lbrack S\rbrack}} & 38\end{matrix}$

where R is ratio A₁/A₂, R_(B)=^(∞)A₁/^(∞)A₂, R_(F)=⁰A₁/⁰A₂, and^(app)K_(d) is an apparent dissociation constant:

$\begin{matrix}{{\,^{app}K_{d}} = {{\frac{0A_{2}}{\infty A_{2}}K_{d}} = {\frac{0A_{2}}{\infty A_{3}}K_{d}}}} & 39\end{matrix}$

2) Cysteine

In some embodiments, the polypeptides as detailed herein comprise atleast one cysteine. As wild-type E. coli GGBP (SEQ ID NO: 55 or 112)does not include any cysteine residues, the polypeptides may include atleast one mutation replacing an amino acid with a cysteine.

In some embodiments, phenylalanine-16 is mutated to or replaced with acysteine (F16C). When the polypeptide consists of a single mutationrelative to wild-type E. coli GGBP (SEQ ID NO: 112), the single mutationis F16C. In some embodiments, tryptophan-183 is mutated to or replacedwith a cysteine (W183C).

In some embodiments, the cysteine provides a thiol for conjugation of areporter group.

b. Reporter Group

The biosenor comprises at least one reporter group conjugated to thepolypeptide. “Reporter” and “reporter group” and “label” are usedinterchangeably herein. The reporter is capable of generating adetectable signal. A variety of reporter groups can be used, differingin the physical nature of signal transduction (e.g., fluorescence,electrochemical, nuclear magnetic resonance (NMR), and electronparamagnetic resonance (EPR)) and in the chemical nature of the reportergroup. In some embodiments, the signal from the reporter is afluorescent signal. Preferably, the reporter group used will form bonds(e.g., thioester bonds) that will remain stable under conditionsrequired for biosensor manufacturing, distribution, and deployment.

The reporter may comprise a fluorophore. Examples of fluorophoresinclude, but are not limited to, acrylodan, badan, rhodamine,naphthalene, danzyl aziridine,4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazoleester (IANBDE),4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole(IANBDA), fluorescein, dipyrrometheneboron difluoride (BODIPY),4-nitrobenzo[c][1,2,5]oxadiazole (NBD), Alexa fluorescent dyes, andderivatives thereof. Fluorescein derivatives may include, for example,5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein,5(6)-carboxyfluorescein, 6-hexachlorofluorescein,6-tetrachlorofluorescein, fluorescein, and isothiocyanate. In someembodiments, the reporter group comprises the thiol-reactive acrylodan(6-acryloyl-2-dimethylaminonaphthalene). In other embodiments, thereporter group comprises thiol-reactive badan(6-bromo-acetyl-2-dimethylamino-naphthalene).

In some embodiments, the reporter is sensitive to its local environmentand exhibits changes in spectral parameters such as intensity and/oremission wavelength depending on factors such as, for example, thedegree of solvent exclusion, and the effective dielectric constant ofthe environment. The fluorescence emission peak and intensity of theacrylodan and badan conjugates are particularly sensitive toconformational changes or ligand binding, making these dyes some of themost useful thiol-reactive probes for reagentless fluorescent biosensorconstruction.

The reporter may be conjugated to a natural amino acid or a naturalamino acid of the polypeptide. The reporter group(s) can be positionedin the glucose-binding pocket of the GGBP as defined by itsthree-dimensional structure (Vyas et al., Nature 1987, 327, 635-638;Vyas et al., Science 1988, 242, 1290-1295; Vyas et al., Biochemistry1994, 33, 4762-4768), so that changes in reporter signal are aconsequence of direct interactions with the bound glucose (endo-stericpositions), at the periphery of the binding site (peristeric positions)where localized changes in the protein structure in response to ligandbinding are sensed, or at some distance way where the reporter group(s)senses glucose binding indirectly via an allosteric coupling mechanism(allosteric positions) (DeLorimier et al., Prot. Sci., 2002, 2655-2675).In some embodiments, the reporter is conjugated to a cysteine of thepolypeptide. In some embodiments, the reporter is covalently conjugatedto the polypeptide via maleimide functional group bound to a cysteine(thiol) on the polypeptide. In some embodiments, the reporter group isattached to a cysteine residue at the endosteric position 183. In otherembodiments, the reporter group is attached to a cysteine residue at theendosteric position 16. In some embodiments, the reporter is conjugatedto F16C. In some embodiments, the reported is conjugated to W183C.

c. Signal

Binding of ligand mediates conformational changes in the biosensor, suchas hinge-bending motions of the polypeptide. The conformational changesaffect the environment of the reporter such that a change in thereporter-generated signal occurs. That is, without ligand bound, thebiosensor results in signal generated from the reporter, and when ligandis bound, the signal generated from the reporter changes. Theligand-bound biosensor results in a reporter-generated signal that isdifferent from the unbound biosensor.

1) Single Wavelength or Range

In some embodiments, the signal comprises the emission intensity of thefluorophore recorded at a single wavelength or range of wavelengths. Thechange in signal may be a shift in the single wavelength or range ofwavelengths. In some embodiments, the shift in the wavelength is atleast about 1 nm, at least about 2 nm, at least about 3 nm, at leastabout 4 nm, at least about 5 nm, at least about 6 nm, at least about 7nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, atleast about 11 nm, at least about 12 nm, at least about 13 nm, at leastabout 14 nm, at least about 15 nm, at least about 16 nm, at least about17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm,at least about 25 nm, at least about 30 nm, at least about 35 nm, atleast about 40 nm, at least about 45 nm, at least about 50 nm, at leastabout 55 nm, at least about 60 nm, at least about 65 nm, at least about70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm,at least about 90 nm, at least about 95 nm, or at least about 100 nm. Insome embodiments, the shift in the wavelength is about 1 nm to about 20nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm toabout 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm,about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm toabout 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, orabout 1 nm to about 10 nm. In some embodiments, the shift in thewavelength is about 1 nm to about 20 nm. In some embodiments, the shiftin the wavelength is about 1 nm to about 100 nm.

2) Two Wavelengths or Ranges

In certain embodiments, the signal comprises the ratio of the emissionintensities recorded at two distinct wavelengths or ranges ofwavelengths. The change in signal may be decreased emission intensity atone wavelength, and no change in emission intensity at the otherwavelength. The change in signal may be increased emission intensity atone wavelength, and no change in emission intensity at the otherwavelength. The change in signal may be increased emission intensity atone wavelength, and increased emission intensity at the otherwavelength. The change in signal may be decreased emission intensity atone wavelength, and decreased emission intensity at the otherwavelength. The change in signal may be increased emission intensity atone wavelength, and decreased emission intensity at the otherwavelength. In some embodiments, the change in ratio of the emissionintensities recorded at two distinct wavelengths or ranges ofwavelengths may be at least about 1.1-fold, at least about 1.2-fold, atleast about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold,at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold,at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold,at least about 5-fold, at least about 5.5-fold, at least about 6-fold,at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold,at least about 8-fold, at least about 8.5-fold, at least about 9-fold,at least about 9.5-fold, at least about 10-fold, at least about 12-fold,at least about 14-fold, at least about 16-fold, at least about 18-fold,at least about 20-fold, at least about 25-fold, at least about 30-fold,at least about 35-fold, at least about 40-fold, at least about 45-fold,at least about 50-fold, at least about 55-fold, at least about 60-fold,at least about 65-fold, at least about 70-fold, at least about 75-fold,at least about 80-fold, at least about 85-fold, at least about 90-fold,at least about 95-fold, or at least about 100-fold.

The change in signal may be a change in the ratio of the two distinctwavelengths or ranges of wavelengths. The change in signal may be ashift in the two distinct wavelengths or ranges of wavelengths. In someembodiments, one wavelength shifts. In some embodiments, bothwavelengths shift. In some embodiments, the shift in the wavelength isat least about 1 nm, at least about 2 nm, at least about 3 nm, at leastabout 4 nm, at least about 5 nm, at least about 6 nm, at least about 7nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, atleast about 11 nm, at least about 12 nm, at least about 13 nm, at leastabout 14 nm, at least about 15 nm, at least about 16 nm, at least about17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm,at least about 25 nm, at least about 30 nm, at least about 35 nm, atleast about 40 nm, at least about 45 nm, at least about 50 nm, at leastabout 55 nm, at least about 60 nm, at least about 65 nm, at least about70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm,at least about 90 nm, at least about 95 nm, or at least about 100 nm. Insome embodiments, the shift in the wavelength is about 1 nm to about 20nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm toabout 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm,about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm toabout 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, orabout 1 nm to about 10 nm. In some embodiments, the shift in thewavelength is about 1 nm to about 20 nm. In some embodiments, the shiftin the wavelength is about 1 nm to about 100 nm.

3. Polynucleotide

Further provided are polynucleotides encoding the polypeptides detailedherein. A vector may include the polynucleotide encoding thepolypeptides detailed herein. To obtain expression of a polypeptide, onetypically subclones the polynucleotide encoding the polypeptide into anexpression vector that contains a promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described(e.g., in Sambrook et al., and Ausubel et al., supra). Bacterialexpression systems for expressing the protein are available in, e.g., E.coli, Bacillus sp., and Salmonella (Paiva et al., Gene 1983, 22,229-235; Mosbach et al., Nature 1983, 302, 543-545). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known inthe art and are also commercially available. Retroviral expressionsystems can be used in the present invention. An example of anexpression vector encoding wild-type E. coli GGBP is shown in FIG. 23 .In some embodiments, the polypeptide comprises an amino acid sequence ofany one of SEQ ID NOs: 1-54. In some embodiments, the polypeptide isencoded by a polynucleotide sequence of any one of SEQ ID NOs: 56-109.

4. Single Biosensor

In some embodiments, the methods and compositions include a plurality ofa single type of biosensor. The biosensors may be identical in structureand function. For example, the biosensors of a single type may have thesame polypeptide, the same reporter, and the same ligand affinity.

5. Panel

In other embodiments, the methods and compositions include a pluralityof different types of biosensors. A plurality of these different typesof biosensors may be arranged or incorporated in a panel. As usedherein, a “panel” refers to two or more biosensors. The two or morebiosensors may be different from each other. The biosensors may differin structure and/or function. Biosensors may differ in polypeptidesequence, reporter, ligand affinities, or a combination thereof.Accordingly, there may be different types of biosensors. In someembodiments, each biosensor in the panel comprises the same reportergroup. In some embodiments, each biosensor in the panel comprises adifferent reporter group. The panel may include at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 30, at least 35, at least 40, at least 45, at least 50, at least55, at least 60, at least 65, at least 70, at least 75, at least 80, atleast 85, at least 90, at least 95, or at least 100 biosensors.

The panel of biosensors includes at least one sensor element. “Sensorelement” refers to a single spot, site, location, or well for the atleast one biosensor, to which a sample or aliquot thereof may beapplied. The panel may be a composite sensor or an array.

a. Composite Sensor

In some embodiments, the panel is a composite sensor. In a compositesensor, each sensor element includes a mixture of two or more differentbiosensors. In some embodiments, the composite sensor includes onesensor element. In some embodiments, the composite sensor includes twoor more sensor elements. In some embodiments, signals are measured froma composite sensor in which the signals arise from one or morebiosensors in the sensor element. For example, signals may be measuredfrom a composite sensor in which the signals arise from a subset of thetotal number of biosensors in the sensor element. For example, signalsmay be measured from a composite sensor in which the signals arise fromtwo of five biosensors in the sensor element.

b. Array

In some embodiments, the panel is an array. In an array, each sensorelement includes a single type of biosensor. An array comprises aplurality of individually and spatially localized sensor elements. Eachsensor element includes a biosensor that is different than or the sameas the biosensor of a different sensor element. In some embodiments,signals are measured from an array in which the signals arise separatelyfrom two or more selected biosensors in separate sensor elements. Anarray may comprise a plurality of sensor elements of a variety of sizesand configurations. An array may comprise a plurality of sensor elementsarranged linearly. For example, an array may comprise a plurality ofmicrometer-sized sensor elements arranged in a single row. An array maycomprise a plurality of sensor elements arranged in a grid. The grid maybe two- or three-dimensional. In some embodiments, the grid is aspatially addressable grid. In some embodiments, the biosensors areincorporated into an array, such as a multichannel or multiplexed array.

6. Administration

The biosensors of the present disclosure can be used in any settingwhere glucose detection is required or desired, such a medical setting(e.g., determining the level of blood glucose in a subject), biologicalsetting (e.g., determining the presence or amount of glucose in areaction), or in process engineering, such as monitoring the amount ofglucose in a fermentation reaction (e.g., beer/wine production, etc.).Other examples include, but are not limited to, uses in the foodindustry (Suleiman et al, In: Biosensor Design and Application:Mathewson and Finley Eds; American Chemical Society, Washington, D.C.1992, vol. 511); in clinical chemistry (Wilkins et al., Med. Eng. Phys.1996, 18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff etal., Endricon 1966, 6, 51-58; Riklin et al., Nature 1995, 376, 672-675);Willner et al., J. Am. Chem. Soc. 1996, 118, 10321-10322); as the basisfor the construction of a fluorescent flow cell containing immobilizedGGBP-FAST conjugates (see, e.g., Wilkins et al., Med. Eng. Phys. 1966,18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff et al.,Endricon. 1966, 6, 51; Group, New Engl. J. Med. 1993, 329, 977-986;Gough et al., Diabetes 1995, 44, 1005-1009); and in an implantabledevices, such as those suitable for use as an artificial pancreas.

The biosensors as detailed herein may be administered in a variety ofways known by those of skill in the art, as appropriate for eachapplication. Biosensors may be provided in a solution. The solution maybe buffered. Biosensors may be provided in a solution and mixed directlywith a sample. Biosensors may be immobilized within a disposablecartridge into which a sample may be introduced or applied. Biosensorsmay be implanted or incorporated in a wearable device. The biosensor maybe provided as an optode.

a. Wearable Device

The biosensor may be attached to or incorporated in a wearable device.Wearable devices may include, for example, adhesive strips, patches, andcontact lenses. The biosensor may be configured for placement in contactwith a subject's skin or mucosal surface. In some embodiments, thebiosensor is configured as an adhesive strip. In some embodiments, thebiosensor is configured within or on the surface of a contact lens. Insome embodiments, the contact lens is formed from a transparentsubstrate shaped to be worn directly over a subject's eye, as describedin, for example, U.S. Pat. No. 8,608,310.

b. Implant

The biosensor may be implanted. The biosensor may be implanted in asubject's body. The biosensor may be implanted in a subject's bloodvessel, vein, eye, natural or artificial pancreas, skin, or anywhere inthe alimentary canal including the stomach, intestine and esophagus. Thebiosensor may be implanted in a subject with a microbead. In someembodiments, the biosensor is configured to be implanted in the skin.The biosensor may be implanted in a subject sub-dermally. The biosensormay generate the signal trans-dermally. In some embodiments, thebiosensor may be implanted in a subject with transdermal microbeads,wherein the optical signals can be transmitted remotely between thebiosensor and detecting device.

c. Optode

In some embodiments, the biosensor is administered as an optode. As usedherein, “optode” refers to an optical fiber with a single biosensor, ora composite biosensor, immobilized at the surface or at the end. An“optode” may also be referred to as an “optrode.” In some embodiments,the biosensor is implanted in a subject as an optode. The optode may beincorporated with or into a needle. The optode may be incorporated witha probe such as endoscopy or colonoscopy probes. The optode may be usedin a tumor, near a tumor, or at the periphery of a tumor. In someembodiments, the biosensor may be implanted in a subject as an optode,wherein the optical signals can be transmitted between the biosensor anddetecting device using physical links. In some embodiments, thebiosensor is administered as an optode to a sample or reaction. Theoptode may be contacted with a sample or reaction. In some embodiments,an optode is used to continuously or episodically monitor a ligand in asample or reaction.

7. Methods

Provided herein are methods of detecting the presence of a ligand,methods of determining the concentration of a ligand, methods ofmonitoring the presence of a ligand, and methods of making a biosensor.The biosensors and methods described herein may be used in conjunctionwith surgery. The biosensors and methods described herein may be used inconjunction with anesthesia. The biosensors and methods described hereinmay be used in conjunction with dialysis. The biosensors and methodsdescribed herein may be used in conjunction with in-line monitoring.In-line monitoring may include removing a sample from a patient,transporting the sample via tubing to a biosensor external to thesubject's body to measure a signal from the biosensor, and returning thesample back to the subject via tubing.

a. Methods of Detecting the Presence of a Ligand

Provided herein is a method of detecting the presence of a ligand in asample. The method may include contacting the biosensor with the sample;measuring a signal from the biosensor; and comparing the signal to aligand-free control. A difference in signal indicates the presence ofligand in the sample.

Provided herein is a method of detecting the presence of glucose in asample. The method may include (a) providing a glucose biosensoraccording to any of the previous claims in which the reporter group isattached the GGBP so that a signal transduced by the reporter group whenthe GGBP is bound to glucose differs from a signal transduced by thereporter group when the GGBP is not bound to glucose; (b) contacting thebiosensor with the test sample under conditions such that the biosensorcan bind to glucose present in the test sample; and (c) comparing thesignal transduced by the reporter group when the biosensor is contactedwith the test sample with the signal transduced by the reporter groupwhen the biosensor is contacted with a glucose-free control sample,wherein a difference in the signal transduced by the reporter group whenthe biosensor is contacted with the test sample, as compared to when thebiosensor is contacted with the control sample, indicates that the testsample contains glucose.

b. Methods of Determining the Concentration of a Ligand

Provided herein is a method of determining the concentration of a ligandin a sample. The method may include contacting the biosensor with thesample; measuring a signal from the biosensor; and comparing the signalto a standard hyperbolic ligand binding curve to determine theconcentration of ligand in the test sample. The standard hyperbolicligand binding curve may be prepared by measuring the signal transducedby the biosensor when contacted with control samples containing knownconcentrations of ligand.

Another aspect of the present disclosure provides a method ofdetermining the concentration of glucose in a test sample comprising,consisting of, or consisting essentially of: (a) providing a glucosebiosensor comprising a glucose biosensor as described herein in whichthe reporter group is attached the GGBP so that a signal transduced bythe reporter group when the GGBP is bound to glucose differs from asignal transduced by the reporter group when the GGBP is not bound toglucose; (b) contacting the biosensor with the test sample underconditions such that the biosensor can bind to glucose present in thetest sample; and (c) comparing the signal transduced by the reportergroup when the biosensor is contacted with the test sample with astandard hyperbolic glucose binding curve prepared by measuring thesignal transduced by the reporter group when the biosensor is contactedwith control samples containing known quantities of glucose to determinethe concentration of glucose in the test sample.

c. Methods of Monitoring the Presence of a Ligand

The present invention is directed to a method of episodically orcontinuously monitoring the presence of a ligand in a reaction. Incertain embodiments, the biosensors may be used in the continuousmonitoring of glucose in a reaction. In certain embodiments, the glucosesensors may be used in episodic monitoring of sample aliquots. Forexample, aliquots of physiological fluids can be analyzed point-of-careor in a laboratory setting.

The method of episodically or continuously monitoring the presence of aligand in a reaction may include contacting the biosensor with thereaction; maintaining the reaction under conditions such that thepolypeptide is capable of binding ligand present in the reaction; andepisodically or continuously monitoring the signal from the biosensor inthe reaction.

The method of episodically or continuously monitoring the presence of aligand in a reaction may include contacting the biosensor with thereaction; maintaining the reaction under conditions such that thepolypeptide is capable of binding ligand present in the reaction;episodically or continuously monitoring the signal from the biosensor inthe reaction; and comparing the signal to a standard hyperbolic ligandbinding curve to determine the concentration of ligand in the testsample. The standard hyperbolic ligand binding curve may be prepared bymeasuring the signal transduced by the biosensor when contacted withcontrol samples containing known concentrations of ligand.

In some embodiments, the method further includes comparing the signal toa ligand-free control, wherein a difference in signal indicates thepresence of ligand in the reaction.

In some embodiments, the method further includes comparing the signal toa standard hyperbolic ligand binding curve to determine theconcentration of ligand in the test sample. The standard hyperbolicligand binding curve may be prepared by measuring the signal transducedby the biosensor when contacted with control samples containing knownconcentrations of ligand.

Another aspect of the present disclosure provides a method ofcontinuously monitoring the presence of glucose in a reactioncomprising, consisting of, or consisting essentially of: (a) providing aglucose biosensor as described herein in which the reporter group isattached the GGBP so that a signal transduced by the reporter group whenthe GGBP is bound to glucose differs from a signal transduced by thereporter group when the GGBP is not bound to glucose; (b) maintainingthe biosensor within the reaction and under conditions such that thebiosensor can bind to glucose present in the reaction; (c) continuouslymonitoring the signal transduced by the reporter group when thebiosensor is contacted with the glucose present in the reaction; andoptionally (d) comparing the signal transduced by the reporter groupwhen the biosensor is contacted with the glucose present in the reactionwith the signal transduced by the reporter group when the biosensor iscontacted with a glucose-free control sample, wherein a difference inthe signal transduced by the reporter group when the biosensor iscontacted with the glucose present in the reaction, as compared to whenthe biosensor is contacted with the control sample, indicates glucose ispresent in the reaction.

Yet another aspect of the present disclosure provides a method ofcontinuously monitoring the concentration of glucose in a reactioncomprising, consisting of, or consisting essentially of: (a) providing aglucose biosensor comprising a glucose biosensor as described herein inwhich the reporter group is attached the GGBP so that a signaltransduced by the reporter group when the GGBP is bound to glucosediffers from a signal transduced by the reporter group when the GGBP isnot bound to glucose; (b) maintaining the biosensor within the reactionunder conditions such that the biosensor can bind to glucose present inthe reaction; and (c) continuously monitoring the signal transduced bythe reporter group when the biosensor is contacted with the glucosepresent in the reaction; and (d) comparing the signal transduced by thereporter group when the biosensor is contacted with the glucose presentin the reaction with a standard hyperbolic glucose binding curveprepared by measuring the signal transduced by the reporter group whenthe biosensor is contacted with control samples containing knownquantities of glucose to determine the concentration of glucose in thereaction.

d. Method of Making a Biosensor

Further provided herein is a method of making a biosensor comprising abinding protein and a fluorophore to form a binding protein-conjugate,the method comprising, consisting of, or consisting essentially of: (1)determining the structure of the binding protein; (2) characterizing thethermodynamic properties of the binding protein-conjugate bound to theligand; (3) generating engineered binding proteins comprising at leastone mutation by performing mutagenesis studies to alter the interactionsbetween the protein and one or more of the fluorophore, direct contactswith the bound ligand, indirect contacts with the bound ligand, andinter-domain contacts that change upon ligand binding; (4) repeating orsuccessively iterating steps (1) and/or (2) and/or (3); and (5)selecting those binding proteins comprising at least one mutation thatexhibit the desired properties for use as a biosensor.

Further provided herein is a method of making a glucose biosensor from aglucose-galactose binding protein comprising, consisting of, orconsisting essentially of: (1) determining the structure of aglucose-galactose binding protein (GGBP); (2) characterizing thethermodynamic properties of the GGBP by determining the fluorescentresponse as a function of glucose concentration, calcium concentration,and temperature; (3) generating GGPBs comprising at least one mutationby performing mutagenesis studies to alter the direct interactionsbetween the protein and fluorophore, direct contacts with bound glucose,indirect contacts with bound glucose, and inter-domain contacts that arepresent in the ligand-bound, but not the ligand-free protein; (4)repeating or successively iterating steps (1) and/or (2) and/or (3); and(5) selecting those glucose binding proteins comprising at least onemutation that exhibit the desired properties for use as a glucosebiosensor.

In some embodiments, the structure of the binding protein may already beknown. In such cases, one may then begin characterizing thethermodynamic properties of the binding protein immediately. In otherembodiments, the structure of the binding protein, e.g., GGBP protein,is determined through any means known to those skilled in the art, suchas X-ray crystallography or Nuclear Magnetic Resonance. In suchembodiments, a three-dimensional coordinate set representing the atomicor near-atomic structure of the fluorescent protein conjugate bound toligand is generated. For example, using such a method, the structuralanalysis of the GGBP183C-acrylodan conjugate glucose biosensor revealedfour essential findings: (1) the structure of amino acid backbone of theGGBP183C-acrylodan in the closed formation is largely indistinguishablefrom that of the wild-type protein bound to glucose; (2) the conjugatedacrylodan points out of the glucose-binding site, vacating the positionthat was occupied by tryptophan 183 in the wild-type protein; (3) in theconjugate, a buried water molecule replaces the tryptophan 183 indolering; (4) acrylodan interacts with protein residues outside the glucosebinding site (5) the salt bridge between residues E149 and K92 in thewild-type protein is broken, resulting in a potentially unfavorableinteraction between the carboxylate of E149 and a carbonyl moiety in theacrylodan conjugate.

For a fluorescent conjugate, a thermodynamic model is developed thatdescribes the dependence of the fluorescence response of the glucosebiosensor to glucose, galactose, calcium, and temperature. Thethermodynamic model is developed to (i) provide a quantitative, preciseand accurate description of the fluorescence landscape (response) as afunction of glucose concentration, calcium concentration, andtemperature; and (ii) provide an accurate description of any potentialsystematic errors in the glucose-dependent signal arising fromvariations in temperature or calcium concentration on pathophysiologicalranges. Development of such a model comprises a means for fluorescentlandscape data collection, such as a Tecan Genesis liquid handlingrobot, that allows for the precise titration series that finely sampledin a geometric (logarithmic) progression, and computer algorithms foranalyzing the data. The data collection means further allows for themeasurement of fluorescence emission intensities of the GGBP-conjugatein the presence of glucose, calcium, etc. and combinations thereof as afunction of temperature.

For example, using such a model on GGBP183C-acrylodan, the modelrevealed several mechanistic features that are relevant to theutilization of some of the glucose biosensors described herein in acontinuous glucose monitoring optrode: (1) the glucose affinity exhibitssignificant temperature dependence; (2) the temperature dependencevaries the glucose concentration relative to that measured at 37° C. ata given signal level by ±3.7% in the diurnal temperature range (36-38°C.) and −7.4% to +18.5% over the pathophysiological range of 35-42° C.;(3) quantitative description of this behavior combined with measurementof the temperature enables high-accuracy sensing; and (4) binding ofCa+2 and glucose are thermodynamically coupled, wherein this effectintroduces a variance in the glucose concentration of ±˜2% at a givensignal and temperature in the 0.8-2.0 mM [Ca+2] concentration range.

Next, the selected glucose biosensors are then affinity tuned bymutagenesis to (i) verify that the wild-type interacts between theprotein and glucose persist in the GGBP conjugate; (ii) establish whichinteractions encode the glucose affinity of the GGBP conjugate; and(iii) identify variants that raise or lower the glucose affinity at adesired temperature to provide the desired characteristics for biosensorfunctionality. Engineered proteins of the present disclosure can beproduced by site-specifically introducing a reporter group(s) by totalsynthesis, semi-synthesis, gene fusions, or reaction of a mutantcontaining a single cysteine with a thiol-reactive reporter group.Introduction of site-specific mutations can be done using methods knownto those skilled in the art. Purification of the glucose biosensors maybe accomplished using numerous means, including fusion with affinitytags placed at the N or C terminal ends, or both. Such methods are wellknown in the art and can be readily determined by one skilled in theart.

EXAMPLES Example 1 Biophysical Analysis and Performance Optimization ofE. coli Glucose-Galactose Binding Protein for Continuous GlucoseMonitoring Executive Summary

An engineered, fluorescently labeled version of glucose-galactosebinding protein, developed originally at Duke University, has beenincorporated successfully into an optrode by Becton-DickinsonTechnologies; this sensor performs well in animal and human trials. Theaims of this project are to gather biophysical information essential forsensor performance analysis, and to further engineer the protein for thedevelopment of next-generation sensors:

(a) To understand the mechanism by which glucose binding is transducedinto a fluorescent signal.(b) To determine the variation in signal response in pathophysiologicaltemperature ranges (35-42° C.) in order to understand potentialtemperature-dependent systematic errors, which is essential forengineering high-accuracy sensors.(c) To determine the variation in signal response in pathophysiologicalcalcium concentration ranges (0.8-1.5 mM), which also is a potentiallyimportant element in determining sensor accuracy.(d) To discover variants that enable the construction ofsecond-generation sensors that provide a near-linear response over awider range of pathophysiological glucose concentrations than ispossible with the current generation sensor based on a single protein.

These aims were all achieved. The high-resolution X-ray structure of thefluorescent acrylodan conjugate with bound glucose revealed interactionsbetween the fluorophore and the protein, and changes in the interactionsbetween the protein and bound glucose. These insights could not havebeen deduced from knowledge of the wild-type structure alone.

Systematic mutagenesis of the glucose-binding site and the interactionsunique to the engineered sensor protein uncovered seven mutations thattune the sensor affinity into the hypo- and hyperglycemic regions. Fourof these mutations involve alterations to interactions that are presentonly in the fluorescent conjugate protein; the structural informationobtained in this effort was therefore useful for their discovery. Thesemutants enable the construction of next-generation, composite sensorswith extended, high-accuracy detection ranges.

A thermodynamic model was developed that accurately describes thefluorescent response of the sensor as a function of glucose and calciumconcentrations and temperature. This analysis clearly reveals that inthe absence of temperature corrections, the error in the glucoseconcentration varies −7.4% to +18.5% over the pathophysiological 35-42°C. temperature range if the sensor were calibrated at 37° C.Determination of the temperature at the sensing tip of thecurrent-generation optrode will eliminate systematic error due to thiseffect. Furthermore, a preliminary analysis of the tuning mutantssuggests that it may be possible to exploit the differences in theirthermodynamic properties to provide the necessary temperaturecorrections in a composite sensor obviating extrinsic thermometry innext-generation, wide range, high accuracy optrodes.

The thermodynamic models also reveal that variation in calciumconcentration influences glucose binding. The systematic error in theglucose concentration uncorrected for calcium concentration is <2% inthe pathophysiological glucose and calcium concentration ranges. Thisfinding establishes that it is not necessary to include an errorcorrection mechanism for calcium concentration in the current generationoptrode. Nevertheless, in next-generation, composite sensor correctionmechanisms to eliminate systematic error due to calcium concentrationfluctuations could be included.

The study reported here therefore has provided critical information forunderstanding and improving current-generation sensor performance.Furthermore, it has provided a route for the development ofnext-generation continuous glucose monitors that incorporate compositesensors to extend coverage in the hypo- and hyperglycemic ranges andfunction with superlative accuracy.

Summary of the Finding

Project Aims: (1) Determine high-resolution structures by X-raycrystallography of the fluorescently labeled glucose-galactose bindingprotein (GGBP) in the presence and absence of glucose and galactose; (2)Construct a detailed thermodynamic characterization of glucose andgalactose affinities as a function of temperature (Gibbs-Helmholtzsurface) and free calcium; (3) Construct variants with affinities in thehyperglycemic glucose concentration range (10-33 mM).

Summary of Achievements

Structure determination: The structure of the GGBP183C⋅acrylodanconjugate glucose complex was determined to 1.5 Å resolution, revealingfour essential findings: (1) The structure of the GGBP183C⋅acrylodanclosed conformation is indistinguishable from wild-type; (2) Theconjugated acrylodan points out of active site, vacating the positionthat was occupied by tryptophan 183 in the wild-type protein; (3) Aburied water molecule replaces the tryptophan 183 indole ring; (4)Acrylodan interacts with protein residues outside the glucose-bindingsite. These unanticipated interactions can be manipulated by mutagenesisfor tuning glucose affinity.

Thermodynamic characterization: A thermodynamic model was developed thataccurately describes the dependence of the fluorescence response ofGGBP183C⋅acrylodan to glucose, galactose, calcium, and temperature. Thismodel revealed several mechanistic features that are relevant to theutilization of GGBP183C⋅acrylodan in a continuous glucose monitoringoptrode: (1) The glucose affinity exhibits significant temperaturedependence, as expected for any chemical equilibrium; (2) Thistemperature dependence varies the glucose concentration relative to thatmeasured at 37° C. at a given signal level by ±3.7% in the diurnaltemperature range (36-38° C.) and −7.4% to +18.5% over thepathophysiological range 35-42° C.; (3) Quantitative description of thisbehavior combined with measurement of the temperature enableshigh-accuracy sensing; (4) Binding of Ca²⁺ and glucose arethermodynamically coupled. This effect introduces a variance in theglucose concentration of ±˜2% at a given signal and temperature in the0.8-2.0 mM [Ca²⁺] concentration range.

Tuning of glucose affinity: Of the 52 mutants examined, seven mutationswere identified that alter the GGBP183C⋅acrylodan glucose affinity,enabling extension of high-accuracy coverage in the hyperglycemic andhypoglycemic concentration ranges by use of a sensor array. Knowledge ofthe three-dimensional structure of GGBP183C⋅acrylodan conjugate wasessential for this effort. The seven mutations divide into three classes(glucose K_(d) values at 37° C. are shown): (1) Interactions with theglucose 6-hydroxyl: N152, K_(d)˜14 mM; F152, K_(d)˜17 mM; Q152, K_(d)˜36mM; (2) Interactions with acrylodan: Q149, S149, K149 all have K_(d)˜0.5mM; (3) Interactions with the buried water molecule: N155, K_(d)˜13 mM.

Materials

Two sets of proteins were studied: the original GGBP183C⋅acrylodanconjugate and its variants (all prepared in this laboratory), and theBD_SM4 variant originally isolated by Becton-Dickinson Technologies,(BDT) and provided to us by BDT as purified, fluorescently labeledprotein.

Fluorescently labeled protein conjugates prepared in this laboratorywere produced by over-expression of a C-terminal hexahistidine-taggedfusion protein, and purified by immobilized metal affinitychromatography and reacted with acrylodan, as described (DeLorimier etal. Prof. Sci. 2002, 11, 2655-2675). Conjugated proteins were stored at4° C. and used for experiments within one month. Point mutants used inthis study were constructed by oligonucleotide-directed mutagenesis, andverified by DNA sequencing.

Structure of GGBP183C⋅Acrylodan Bound to Glucose

Structure determination: Crystallization conditions for theGGBP183C⋅acrylodan conjugate were determined using 0.5 μL sitting drops(0.1 mM protein, 125 mM glucose, 1 mM CaCl₂), 10 mM KCl, 10 mM MOPS, pH7.4) in five 96-well sparse-matrix screens stored at 4° C. and 17° C.,using a Mosquito liquid-handling robot. The most promising hits wererefined in focused screens set up by hand, exploring PEG lengths andconcentrations, and pH; a 96-well additive screen was also explored withautomation, but yielded no improvements.

Diffraction-quality crystals were grown in 5 μL sitting drops at 4° C.two weeks (26% PEG 4,000, pH 7.75). A similar screen using BD_SM4 didnot yield any crystals. We postulate that the additional purificationand immobilization tags present in this protein inhibitedcrystallization. We were also unable to obtain crystals ofGGBP183C⋅acrylodan in the absence of glucose.

After transfer to a cryprotectant solution containing ˜2.5 M glucose,crystals were flash-frozen in liquid nitrogen. Diffraction data wascollected at 100K at the Advanced Photon Source, Beamline 22-BM (ArgonneNational Laboratory). The structure was solved to 1.5 A resolution bymolecular replacement using 2GBP as the search model. Structurerefinement is currently being finished in preparation for publication.

Analysis: The overall structure of the GGBP183C⋅acrylodan glucosecomplex is almost indistinguishable from the wild-type closed formconformation (FIG. 1 ). Acrylodan is attached covalently to the W183Cmutation, which is located adjacent to the bound glucose in the interiorof the closed conformation. The conjugated acrylodan ring system hasvacated the position occupied by the tryptophan indole ring in thewild-type protein, and has swung out of the binding pocket, pointinginto the solvent. The vacated ring position is replaced by a watermolecule buried adjacent to the bound glucose (FIG. 2 ). This water iscontacted by alanine 155, which in the wild-type protein is locatedadjacent to the W183 indole ring. The protein backbone barely adjusts toaccommodate the acrylodan position. However, glutamate 149 and lysine 92move, opening an aperture to the surface through which the acrylodanfits. This motion breaks a wild-type, inter-domain salt bridge betweenE149 and K92, and places the carboxylate of E149 in close proximity tothe acrylodan carbonyl, which may represent an unfavorable contact.Other than the replacement W183C indole ring with water, theinteractions between the protein and the bound glucose have not changed.

These findings establish that the effects of introducing the acrylodanconjugate are remarkably localized. The 10,000-fold decrease in theaffinity of the GGBP183C⋅acrylodan for glucose compared to the wild-typeprotein is therefore a consequence of the loss of the interaction withthe W183 indole ring, the loss of the inter-domain salt bridge,unfavorable interactions between the surface residues contacting theacrylodan, or a combination of these effects. These structuralhypotheses were tested by mutagenesis (see below).

Thermodynamic Characterization

A detailed thermodynamic model of the sensor response was developed to(i) provide a quantitative, accurate description of the fluorescencelandscape (response) as a function of glucose concentration, calciumconcentration, and temperature; and (ii) provide an accurate descriptionof any potential systematic errors in the glucose-dependent signalarising from variations in temperature or calcium concentration inpathophysiological ranges.

Fluorescent Landscape Data Collection

As a first step, we developed automated liquid-handling methods toconstruct precise, titration series that are finely sampled in ageometric (logarithmic) progression. This required designing theappropriate configuration of a programmable liquid-handling robot (FIG.3 ), and writing a software environment that translates an abstract,multi-component titration series into machine instructions to operatethe robot (FIG. 4 ).

The titration series were mixed with GGBP183C⋅acrylodan orBD_SM4⋅acrylodan conjugates in 384-well microtiter plates. Thetemperature dependence of the titrations was measured in a real-time PCRmachine (Roche LightCycler 480) by determining the ratio of fluorescenceemission intensities at 488, 510, and 580 nm (excitation at 440 nm) as afunction of temperature (290-370K). Temperatures were advanced in 1Ksteps. At each temperature data was collected for at 1-second intervalsfor 60 seconds at which point the signal had relaxed to a steady valueassociated with the new temperature. Collection times were adjusted tominimize photobleaching (which was not detectable under theseexperimental conditions). Data reduction software was developed toconvert these observations into time-independent datasets that recordfluorescence as a function of temperature for each well and associatewells with their concentration of titrant and additive. Management toolswere developed to maintain a database of titrations and their analyses.

Three-dimensional landscapes of fluorescence recorded as a function ofglucose concentration and temperature were collected at eight differentcalcium concentrations. In addition, we collected the fluorescentlandscape in a calcium titration determined in the absence of glucose.

Thermodynamic Models of the Fluorescent Landscapes

FIG. 5 shows the dependence of the fluorescent signal on glucoseconcentration and temperature at 1 mM Ca²⁺ for the GGBP183C⋅acrylodanand BD_SM4⋅Acrylodan conjugates. Considerable effort was expended indeveloping the software necessary for quantitative modeling of thefluorescence response.

Extensive analysis of a variety of quantitative model revealed that thechanges in fluorescence arise from temperature- and ligand-dependentchanges between various states of the system, each of which isassociated with a fluorescence base plane. The total fluorescence signal(i.e., the landscape) as a function of temperature T and ligandconcentration L, s(T,L), is then given by

$\begin{matrix}{{s\left( {T,L} \right)} = {\sum\limits_{i}{{\beta\left( {T,L} \right)}_{i}{f\left( {T,L} \right)}_{i}}}} & 1\end{matrix}$

where β_(i) is the fluorescence base plane associated with the fractionof the ith state fi. Each base plane is defined by its lineartemperature dependence on temperature and ligand concentration:

$\begin{matrix}{\beta_{i} = {s_{0,i} + \left( \frac{\partial S}{\partial T} \right)_{i} + {\sum\limits_{j}\left( \frac{\partial S}{\partial T} \right)_{i}}}} & 2\end{matrix}$

where S_(0,i) is the signal at 0 K in the absence of ligand,

$\left( \frac{\partial S}{\partial T} \right)_{i}\left( \frac{\partial S}{\partial T} \right)_{i}{and}\left( \frac{\partial S}{\partial L_{j}} \right)_{i}\left( \frac{\partial S}{\partial L_{j}} \right)_{i}$

the partial derivatives with respect to temperature and the j^(th)ligand species respectively.

The two landscapes shown in in FIG. 5 can be described in terms of threemajor states: the folded apo protein (A), the folded saturated glucosecomplex (S), and the denatured state (D). The signal decreases both inresponse to glucose binding, and thermal denaturation. In both proteins,the denatured state is split into two sub-states, D₁ and D₂, by aconformational transition that occurs at high temperatures (˜360 K). InGGBP183C⋅acrylodan the glucose complex undergoes a conformational changeat ˜300 K, splitting into sub-states S1 and S2. The signal “ridge” thatseparates S and D at high glucose concentrations and elevatedtemperatures (320-340 K) is the consequence of two effects. First, asthe temperature increases, the affinity for glucose is lowered, andapo-protein forms, which has a higher signal than the complex: the ridgerises. Second, as the temperature increases further, the proteinthermally denatures, and the baseplane of the denatured statepredominates: the ridge drops. It is also readily apparent that thestability of the protein increases significantly in this region: themid-point of the ridge moves to higher temperatures with increasedglucose concentration. This is the consequence of two other effects.First, ligand binding stabilizes the folded state. Second, glucose is astabilizing osmolyte. Such reagents alter protein stability by changingthe water activity, typically at high concentrations. Their effect onfree energy is linear with osmolyte concentration. The glucose osmolyteeffect dominates in this region (see the linear dependence on the ridgemid-point in FIG. 5C and FIG. 5F). These relationships are depicted inFIG. 6 .

(1) Thermal unfolding converts the folded state(s), N, into the unfoldedstate(s), D.The free energy of unfolding ΔG_(U)(T,L) is dependent on

a. The temperature dependence of thermal stability in the absence of anyligand, Δ^(apo)G_(U)(T).

b. Free energy of binding of glucose to the folded state,ΔG_(b,glc)(T,L).

c. Free energy of binding Ca²⁺ to both the folded, Δ^(N)G_(b,ca)(T,L),and denatured states, Δ^(D)G_(b,ca)(T,L).

d. The free energy of the osmolyte effect of glucose on the free energyof the folded apo-protein Δ^(N)G_(o).

(2) In both proteins, the unfolded state undergoes a conformationalchange, splitting it into two sub-states, D₁ and D₂. This free energy,Δ^(D)G_(C)(T) is dependent on temperature.

(3) In both proteins glucose binds to the folded state, convertingbetween the apo state, A, and saturated ligand complex, S. This freeenergy, ΔG_(b,glc)(T L), is dependent on glucose concentration andtemperature.

(4) In GGBP183C⋅acrylodan the saturated glucose complex undergoes aconformational transition that further splits it into two states, S₁ andS₂. The free energy of this process Δ^(S)Gc(T), is dependent ontemperature and the osmolyte effect of glucose on conformationaltransitions λ^(C)G_(O).

This system is described by the following set of nested equations.

$\begin{matrix}{{\Delta{G_{U}\left( {T,L} \right)}} = {{\Delta^{apo}{G_{U}(T)}} - {\Delta{G_{b,{glc}}\left( {T,L} \right)}} + {\Delta^{D}{G_{b,{Ca}}\left( {T,L} \right)}} - {\Delta^{N}{G_{b,{Ca}}\left( {T,L} \right)}} + {\Delta^{N}G_{o}}}} & 3\end{matrix}$ $\begin{matrix}{{\Delta^{apo}{G_{U}(T)}} = {{\Delta H}_{U}^{\cdot} + {\Delta{C_{p,u}\left( {T - T_{m}} \right)}} - {T\left( {\frac{\Delta H_{U}^{\cdot}}{T_{m}} + {\Delta C_{p,u}\ln\frac{T}{T_{m}}}} \right)}}} & 4\end{matrix}$

is the Gibbs-Helmholtz description of the temperature dependence ofunfolding, where T_(m) is the mid-point of the thermal transition whereΔ^(apo)G_(U)(T)=0, ΔH_(U), the enthalpy at the T_(m), and ΔC_(p,u) theheat capacity.

ΔG _(b,x)(T,L)=−RT ln Q _(b,X)(T,L)  5

is the binding energy of the binding polynomial for ligand X (glucose orCa²⁺).

$\begin{matrix}{{Q_{b,X}\left( {T,L} \right)} = {1 + \frac{L}{K_{d,X}(T)}}} & 6\end{matrix}$

is the binding polynomial for a single ligand-binding site, whereK_(d,X)(T) is the temperature-dependent dissociation constant for ligandX.

$\begin{matrix}{K_{d,X} = \left( {e^{{- \Delta}G_{b,X}^{\cdot}/{RT}} - 1} \right)^{- 1}} & 7\end{matrix}$ $\begin{matrix}{{\Delta G_{b,X}^{\cdot}} = {{\Delta H_{b,x}^{\cdot}} + {\Delta{C_{p,b,X}\left( {T - T^{*}} \right)}} - {T\left( {\frac{\Delta H_{b,X}^{\cdot}}{T^{\cdot}} + {\Delta C_{p,b,X}\ln\frac{T}{T^{\cdot}}}} \right)}}} & 8\end{matrix}$

is the Gibbs-Helmholtz description of the free energy of binding ligandX under standard state conditions ([X]=1 M), where T* is the temperatureat which ΔG_(b,X)=0, ΔH_(b,X) the standard binding enthalpy at T*, andΔC_(p,b,X) the ligand-binding reaction heat capacity.

Δ^(N) G _(O) =m _(N)[glucose]

is the linear free energy relationship that arises from the osmolyteeffect due to glucose.

$\begin{matrix}{{\Delta{G_{C}(T)}} = {{\Delta H_{C}^{\cdot}} + {\Delta C_{p,c}} + {\Delta{C_{p,c}\left( {T - T_{c}} \right)}} - {T\left( {\frac{\Delta H_{C}^{\cdot}}{T_{C}} + {\Delta C_{p,c}\ln\frac{T}{T_{C}}}} \right)} + {\Delta^{C}G_{O}}}} & 9\end{matrix}$

is the Gibbs-Helmholtz description of the temperature dependence of atwo-state conformational change (in the denatured state of the saturatedglucose complex).

Δ^(C) G _(O) =m _(c)[glucose]

Landscape equation 1 expands as

S(T,L)=f _(N)(1− y _(glc))β_(A) +f _(N) y _(glc)β_(S)+(1−f _(N))f_(D1)β_(D1)+(1−f _(N))(1−f _(D1))β_(D2)  10

For BD_SM4-acrylodan, and

S(T,L)=f _(N)(1− y _(glc))β_(A) +f _(N) y _(glc) f _(S1)β_(S1) +f _(N) y_(glc)(1−f _(S1))β_(S1)+(1−f _(N))f _(D1)β_(D1)+(1−f _(n))(1−f_(D1))β_(D2)  11

For GBP183C⋅Acrylodan. To get the fractions of each state:

$\begin{matrix}{f_{N} = {\frac{1}{1 + {K_{U}\left( {T,L} \right)}}{and}{K_{U}\left( {T,{L = {e\frac{\Delta{G_{U}\left( {T,L} \right)}}{RT}}}} \right.}}} & 12\end{matrix}$ $\begin{matrix}{f_{D1} = {{\frac{1}{1 + {{\,^{D}K_{C}}(T)}}{and}{\,^{D}K_{C}}(T)} = {e\frac{\Delta{\,^{D}G_{C}}(T)}{RT}}}} & 13\end{matrix}$ $\begin{matrix}{f_{S1} = {{\frac{1}{1 + {{\,^{S}K_{C}}(T)}}{and}{\,^{S}K_{C}}(T)} = {e\frac{\Delta{\,^{S}G_{C}}(T)}{RT}}}} & 14\end{matrix}$ $\begin{matrix}{{\overset{\_}{y}}_{glc} = \frac{1}{1 + \frac{\lbrack{glc}\rbrack}{K_{d,{glc}}(T)}}} & 15\end{matrix}$

Fits of these equation systems to experimental observations such asshown in FIG. 5 require good initial estimates of the parameters. Thesecan then be refined using multi-dimensional conjugate gradient methods.To obtain local fits for protein thermostability in the absence ofligand we solve

s(T)=f _(N)β_(A)+(1−f _(N))β_(D)  16

using 12, with the van't Hoff approximation:

$\begin{matrix}{{K_{U}(T)} = {e\frac{\Delta{H_{U}^{\cdot}\left( {\frac{1}{T} - \frac{1}{T_{m}}} \right)}}{R}}} & 17\end{matrix}$

This gives excellent fits (FIG. 7 ).

The response of acrylodan to glucose binding was separated from theeffects of thermal denaturation by extracting binding isotherms withinthe temperature range where the apo-protein remains >99% folded (T<320K; FIG. 7 ). Within this temperature range, binding isotherms wereconstructed by extracting fluorescence values across different wells,each containing different glucose concentrations, at a giventemperature, and fit individually with high accuracy to isotherms (FIG.8 ):

S _(T)(L)= y _(T) ^(S)β_(T)+(1− y _(T))^(A)β_(T)  18

using linear baselines which vary individually in accordance with thetilt of the S, S₁, and S₂ baseplanes that correspond the saturatedglucose complex. The individually fit K_(d)(T) values were convertedinto ΔG_(b)(T) and fit to the Gibbs-Helmholtz equation 9 (FIG. 9 ).

The thermally-driven conformational change in the saturated glucosecomplex of GBP183C⋅Acrylodan (FIG. 10 ) was fit to a two-state modelwith linear baselines using equations 16 and 17 within temperatureranges where the fluorescence signal is expected to be dominated by thiseffect (FIG. 10A). These thermal transitions were fit individually forwells containing glucose concentrations in excess of 0.2 M. Theresulting values for transition mid-point temperatures, T_(c), andconformational enthalpy, ΔH_(c), fit to a linear dependence on glucoseconcentration (FIG. 10B), consistent with an osmolyte effect.

The initial values obtained by the local fits were used to obtain aglobal fit of the dependence of the fluorescent signal on glucoseconcentration and temperature at 1 mM CaCl₂) using conjugate gradientminimization of least squares difference between the object functiondescribed by equations 1-15 and the experimental observations (FIG. 11). Bootstrap analysis (randomly duplicating 25% of the experimentaldatapoints and resolving from the initial conditions) indicated that thesolution is stable.

GGBP binds Ca²⁺ at a location remote from glucose (FIG. 1 ). The bindingof Ca²⁺ was investigated in the absence of glucose (FIG. 12 ). Thefluorescence of acrylodan does not respond directly to Ca²⁺ binding,unlike glucose. Nevertheless, Ca²⁺ binding can be followed through itseffect on protein stability (FIG. 13 ). Low Ca²⁺ concentrations raisethe stability of the protein significantly, indicating the presence of ahigh-affinity site in the native state, consistent with the proteinstructure. However, at elevated concentrations, this stabilizationeffect levels off and addition of Ca²⁺ becomes destabilizing. Thisbehavior indicates that the denatured state has multiple, low-affinityCa²⁺ binding sites.

These effects can be modeled accurately by a fit of the ligand- andtemperature-dependent free energy of stability for Ca²⁺ (Judge et al.Diabetes Technol. Thera. 2011, 13, 309-317; Layton et al. Biochemistry2010, 49, 10831-10841).

ΔG _(U)(T,L)=Δ^(apo) G _(U)(T)+Δ^(D) G _(b,Ca)(T,L)−Δ^(N) G_(b,Ca)(T,L)+Δ^(apo) G _(O)  19

The individual terms are cast in terms of T and [Ca²⁺] using equations4-9. However, rather than fitting fluorescent landscape data directlyusing 1, T_(m) and ΔH_(u), values are extracted at each Ca²⁺concentration using 16 and 17, and fit to the equation system bydetermining the root of 19 to obtain the calculated T_(m) value. Therelationship

ΔH _(U) ^(⋅)(T,L)=Δ^(apo) H _(U) ^(⋅)+Δ^(D) H _(b)(T,L)−Δ^(N) H_(b)(T,L)  20

was used to obtain the calculated enthalpy at a given Ca²⁺concentration, where

ΔH _(b)(T,L)= y (ΔH _(b) ^(⋅) +ΔC _(p,b)(T−T ^(⋅)))  21

This analysis reveals that in the absence of glucose bothGGBP183C⋅acrylodan and BD_SM4⋅acrylodan conjugates bind Ca²⁺ at 37° C.with ˜10 nM affinity in the native state at a single site, consistentwith the protein structure, and have three low-affinity (ranging 0.4-12mM) Ca²⁺-binding sites in the denatured state.

To investigate whether the glucose- and Ca²⁺-binding sites interact inthe folded state, fluorescent landscapes of glucose titrations werecollected at eight different calcium concentrations. Each of theselandscapes was fit individually to equation 1. From these fits thedependence of glucose binding on calcium could be determined (FIG. 14 ).Glucose affinity and binding and folding heat capacities are linked tocalcium binding.

The dependence of calcium binding on glucose concentration also can beextracted from these landscapes, by fitting equation 19 to thermal meltsat obtained at a particular glucose concentration (FIG. 15 ). Thisanalysis reveals that Ca²⁺ binding to the native state also is linked tothe glucose binding, as would be expected.

Taken together, the landscapes reveal a complex set of interactionslinking the binding of the two ligands to each other and to proteinstability, summarized in FIG. 16 . In this model, the folded state hastwo conformations, N₁ and N₂, which differ in their affinities forglucose and Ca²⁺ and in their folding properties. Compared to N₂, the N₁state has lower affinity for glucose and Ca²⁺ and a lower ΔC_(p,u). Thisis consistent with the domain containing the Ca²⁺ site being partiallyunfolded in the absence of ligand. ΔC_(p,u) is proportional to thedifference in the solvent-accessible surface are in the unfolded andfolded states. Accordingly, a form that has a partially unfolded statehas a smaller heat capacity compared to a protein that adopts morestructure. A partially unfolded state has been observed for GGBP bycircular dichroism. In the absence of ligand N₁ is favored over N₂. Aseither ligand binds, the equilibrium shifts towards N₂, therebyincreasing the affinity for the other ligand (this is a classicheterotropic cooperative binding effect), and altering the apparentthermal denaturation behavior. The equilibrium between N₁ and N₂ isdifferent in GBP183C⋅acrylodan and BD_SM4⋅acrylodan: in the latter, N₁is more favored, requiring higher concentrations of Ca²⁺ or glucose toshift to N₂. In GGBP183C⋅acrylodan, the glucose-bound state of N₁,undergoes a further thermally driven conformational transition,associated with a change in fluorescence.

The unfolded state has three Ca²⁺ binding sites that differ in affinity,but no glucose-binding site. The effect of Ca²⁺ binding to the unfoldedstate is seen at elevated concentrations. This effect destabilizes theprotein above ˜3 mM calcium. The effect of Ca²⁺ binding on ΔC_(p,u) isconsistent with this model: at low concentrations, it organizes thepartially unfolded state, increasing ΔC_(p,u) at elevatedconcentrations, it organizes the unfolded state, decreasing ΔC_(p,u)

These effects can be combined into one unified thermodynamic model thatdescribes the four-dimensional surface s(T,[glucose],[Ca²⁺]). This modelis currently being worked on.

Analysis of Systematic Errors

At a given glucose concentration, the signal is sensitive to temperatureand calcium concentration. Consequently a systematic error can beintroduced in glucose measurements, if these factors are not taken intoaccount. The thermodynamic model described above can be used to providea quantitative analysis of such potential systematic errors bydetermining how the glucose concentration varies with temperature atconstant signal: i.e., the error is determined by the temperaturedependence of the isochrome (FIG. 17 ). The temperature dependent errorfor the variation shown in the figure is:

$\begin{matrix}{{\varepsilon(T)} = \frac{L_{true} - L_{cal}}{L_{cal}}} & 22\end{matrix}$

This error function can be calculated from the thermodynamic models(FIG. 18 ). As can be seen, this systematic error is quite significantε(T)˜3.7% for each degree away from the calibration temperature forBD_SM4⋅acrylodan. This means that in the diurnal range of 36° C.-37° C.,the error is ±3.7% (7.4% range), whereas in the full pathophysiologicalrange of 35° C.-42° C. it ranges from −7.4% to +18.5% (25.9% range).

The linkage between glucose and Ca²⁺ binding also introduces asystematic error in a sensor calibrated at a given calciumconcentration. However, in the 0.8-1.5 mM pathophysiological calciumconcentration range, this effect is relatively small. The full-scalethermodynamic model is not yet complete, so we cannot calculate thiserror precisely. However, the variation of the isothermal K_(d) forglucose at 37° C. is approximately 4.7±0.1 mM (FIG. 14 ), correspondingto a 2% error.

Affinity Tuning

Mutagenesis studies were carried out to (i) verify that the wild-typeinteractions between the protein and glucose persist in theGGBP183C⋅acrylodan conjugate; (ii) establish which interactions encodethe dramatic lowering of glucose affinity of the GGBP183C⋅acrylodanconjugate; and (iii) identify variants that raise the glucose affinityat 37° C. to provide improved coverage of the response in thehyperglycemic region (10-33 mM), and lower it to improve coverage thehypoglycemic region (2-4 mM).

Analysis of mutant affinity values: Isothermal glucose titrations wereextracted from the fluorescent landscape or emission datasets asdescribed above. Monochromatic emission intensities I_(λ). (theseintensities correspond to a bandpass intensity recorded with a physicalfilter in the case of the Roche LightCycler) were fit to

I _(λ)=^(apo)β_(λ)(1− y _(true))+^(sat)β_(λ) y _(true)  23

Where ^(apo)β_(λ) and ^(sat)β_(λ) are the fluorescence baselinesassociated with the ligand-free and ligand-bound states of the protein,respectively, and y _(true) y _(true) the fractional saturation of theprotein. Baseline functions can be constant, linear, or a second-orderpolynomial. For the ligand- and temperature-dependent fluorescencelandscapes, we use a constant value for ^(apo)β_(λ), but ^(sat)β_(λ) isdescribed by a linear dependence on glucose concentration, [L]:

^(apo)β_(λ) =a _(x) +b _(x)[L]β_(λ) =a _(x) +b _(x)[L]  24

For a single glucose-binding site, the fractional saturation is given by

$\begin{matrix}{\overset{\_}{y} = \frac{\lbrack L\rbrack}{\lbrack L\rbrack + K_{d}}} & 25\end{matrix}$

where [L] is the ligand (glucose) concentration and K_(d) thedissociation constant, ^(true)K_(d) ^(true)K_(d) for y _(true),y_(true).A dichromatic ratiometric signal is defined as the ratio of theintensities at two independent wavelengths, λ₁ and λ₂λ₁ and λ₂

R _(1,1) =I _(λ1) /I _(λ2)  26

This signal removes systematic error due to variations in conjugateconcentration, and fluctuations in excitation source intensities, anddetector-dependent changes in emission intensities. It is a key aspectfor high-precision sensing using the reagentlessfluorescently-responsive sensors described here. The ratiometric signalalso can be fit to a binding isotherm:

R _(1,2)=^(apo)β_(R)(1− y _(R))+^(sat)β_(R) y _(R)  27

where ^(apo)β_(R) and ^(sat)β_(R) are the baselines, and y _(R) y _(R)the apparent fractional saturation of the protein (with ^(app)K_(d)^(app)K_(d)). In general, ^(true)K_(d) ^(true)K_(d)≠^(app)K_(d)^(app)K_(d); if both baselines are constant, a simple relationship canbe derived relating ^(app)K_(d) ^(app)K_(d) to ^(true)K_(d)^(true)K_(d):

$\begin{matrix}{{\,^{app}K_{d}} = {{\,^{true}K_{d}}\frac{\,^{apo}I_{\lambda 2}}{\,^{sat}I_{\lambda 2}}}} & 28\end{matrix}$

where ^(apo)I_(λ2) ^(apo)I_(λ2) and ^(sat)I_(λ2) ^(sat)I_(λ2) are theemission intensities of the monochromatic signal at wavelength λ₂λ₂ ofthe ligand-free and ligand-bound protein, respectively. Equation 28illustrates that the ^(app)K_(d) ^(app)K_(d) values obtained by fits of25 to ratiometric signals are wavelength dependent. To obtainwavelength-independent values needed for comparison of mutant proteins,it is necessary to simultaneously fit ^(app)K_(d) ^(app)K_(d) to theratiometric signal and ^(true)K_(d) ^(true)K_(d) to the twomonochromatic signals. For a given isothermal titration, values for^(app)K_(d) ^(app)K_(d) and ^(true)K_(d) ^(true)K_(d) were obtainedusing a non-linear fitting algorithm in which these two parameters weresimultaneously fit to the three experimental binding isotherms usingequations 23 and 27, with the two monochromatic isotherms sharing thesame ^(true)K_(d) ^(true)K_(d) value. Three separate pairs of linear^(apo)β ^(apo)β and ^(sat)β ^(sat)β baselines were fit in thisprocedure. TABLES 1-5 record the ^(true)K_(d) ^(true)K_(d) values at 25°C., obtained by analysis of Roche LightCycler data in 488 nm and 510 nmchannels. The uncertainty of the fit (‘error’) was obtained using aboot-strapping technique in which 37% of the data was replicated. In allcases, no binding was recorded if one of ^(app)K_(d) ^(app)K_(d) or^(true)K_(d) ^(true)K_(d) exceeded 200 mM. The glucose constant ofGGBP183 C-acrylodan is 4.21±0.08 mM at 25° C.

Alanine-scanning mutagenesis: The major, direct interactions betweenbound glucose and the protein (FIG. 2 ) were probed by alanine-scanningmutagenesis (TABLE 1). Loss of these interactions individually leads tosignificant loss in affinity, consistent with their hydrogen bondformation to the glucose hydroxyls, and van der Waals interactions withits pyranose ring.

TABLE 1 Alanine scanning mutagenesis. Description of wild-type residueMutation K_(d) ^(298K)(glucose)/mM T_(m)/K position Y10A No binding 326Interacts with D14 across the mouth of the binding site. Also makescontact with Acrylodan. D14A 174 ± 96 317 Hydrogen bond with glucose-OH4. Also makes this contact with galactose. F16A No binding 319 Part ofthe aromatic sandwich. N91A No binding 326 Hydrogen bond to glucose-O6hydroxyl. Weak contact with glucose-O5 (pyranose ring oxygen). K92A Nobinding 323 In the wild-type protein it interacts with Glu149 across themouth of the protein. H152A No binding 316 Hydrogen bond to glucose*O6hydroxyl. May make contact with the Acrylodan. D154A No binding 320Hydrogen bond to glucose-O1. R158A No binding 322 Hydrogen bonds toglucose-O1 and —O2. N211A No binding 317 Hydrogen bond to glucose-O3.D236A No binding 320 Hydrogen bonds to glucose-O2 and —O3. N256A Nobinding 324 Weak hydrogen bonds to glucose- O1 and —O2.

Affinity Tuning

Interactions with the buried water molecule that replaces W183:A155mutants: The methyl group of alanine 155 points towards a buried watermolecule that replaces the W183 indole ring in the GGBP183C⋅acrylodanconjugate (FIG. 19 ). In the wild-type protein, A155 is adjacent to theface of the W183 indole ring that does not touch the bound glucose (FIG.19 ). We constructed a series of mutations to test whether it ispossible for amino acid side-chains placed at position 155 to form a vander Waals contact between the protein and the glucose pyranose ringwithout interfering with the acrylodan conjugate attached to 183C (TABLE2).

We also identified a variant at this position, A155N, which has theinteresting property that glucose binding has the opposite effect onfluorescence: it increases the 488/580 ratio. Binding of glucosetherefore shifts the emission wavelength maximum to the blue instead ofthe red.

TABLE 2 Mutations at position 155. Mutation K_(d) ^(298K)(glucose)/mMT_(m)/K A155S No binding 321 A155H 13.5 ± 2.5  314 A155L No binding 320A155F No binding 321 A155Y No binding 321 A155N 5.9 ± 1.0 320 A155K Nobinding 318 A155M No binding 321 A155W No binding 322 A155Q No binding321

The effect of potentially unfavorable contacts with acrylodan: E149mutants: The E149 carboxylate is in close proximity to the acrylodancarbonyl (FIG. 19 ). We constructed three variants to remove or reversethis charge (TABLE 3). All three variants improved the glucose affinityconsistent with the removal of an unfavorable interaction. Furthermore,these higher affinity mutants extend the coverage into the hypoglycemicrange—a desirable achievement that exceeds the specification in theoriginal Aims.

TABLE 3 Mutations at position 149. Mutation K_(d) ^(298K)(glucose)/mMT_(m)/K E149Q 0.46 ± 0.01 322 E149S 0.34 ± 0.01 320 E149K 1.44 ± 0.04321

Tuning affinity by altering direct interactions with glucose:Construction of variants with affinities in the 10-33 mM range requiresthat interactions are perturbed only minimally (2-5 fold loss ininteraction strength). We explored whether it is possible to achievesuch subtle changes through alteration of individual hydrogen bond orvan der Waals contact strengths by mutagenesis at eight positions thatform direct interactions with glucose (FIG. 2 ; TABLE 4). Three of the34 mutants that were tried yielded the desired result (H152N, H152Q, andH152F). All three involve changes in histidine 152, which forms ahydrogen bond through N, with the glucose 6-hydroxyl (FIG. 19 ). Thesethree mutants cover the range specified in the original Aims.

TABLE 4 Tuning affinity by altering interactions with glucose. MutationK_(d) ^(298K)(glucose)/mM T_(m)/K Description D14E No binding 318Hydrogen bonds with glucose- OH4. Also makes this contact withgalactose. D14Q No binding D14N No binding 317 D14S No binding D14T Nobinding 316 D14H No binding 317 D14L No binding 311 D14Y No binding D14FNo binding F16L No binding 321 Part of the aromatic sandwich. D154N Nobinding 318 Hydrogen bonds to glucose-O1. R158K No binding 318 Hydrogenbonds to glucose-O1 and —O2. D236N No binding 320 Hydrogen bonds toglucose-O2 and —O3. N256D No binding 321 Weak hydrogen bonds to glucose-O1 and —O2. H152F  21.1 ± 0.9. 321 Hydrogen bonds to glucose-O6hydroxyl. May make contact with acrylodan. H152Q 25.8 ± 2.0 322 H152N18.8 ± 1.3 322 H152K No binding 319 N211Y No binding 316 Hydrogen bondsto glucose-O3. N211F No binding 323 N211W No binding 328 N211K Nobinding 315 N211Q No binding 317 N211S No binding 319 N211H No binding320 N211M No binding 317 M182W No binding 322 Adjacent to acrylodan.

Specificity

The galactose affinities for those mutants that bind glucose were alsodetermined.

TABLE 5 Galactose affinities. Mutation K_(d) ^(298K)(glucose)/mM K_(d)^(298K)(galactose)/mM D14A 174 ± 96   90 ± 21 E149Q 0.46 ± 0.01 10.8 ±0.4 E149S 0.34 ± 0.01 12.7 ± 15  E149K 1.44 ± 0.04 40.2 ± 1.3 H152F 21.1± 0.9  No binding H152N 18.8 ± 1.3  No binding H152Q 25.8 ± 2.0  Nobinding A155H 13.5 ± 2.5  17.7 ± 2.1  A155N 5.9 ± 1.0 7.5 ± 1.0

Extending the Detection Range in a Composite Sensor

Seven proteins have been identified that extend the detection range intothe hypo and hyperglycemic regions. Each of the sensors exhibits adistinct fluorescent landscape (FIG. 20 ). Their isochromes have uniquetemperature signatures that arise from individual temperaturedependencies on glucose binding, conformational changes, and baseplanes.This means that, in principle, temperature correction can be achieved byanalyzing the signals of several sensors in combination.

A minimum of three can be combined with the current sensor to constructa four-component composite sensor has a significantly extendedhigh-accuracy detection range (FIG. 21 ).

Example 2 Properties of the F16C Endosteric Mutation of E. coliGlucose-Galactose Binding Protein

The acrylodan conjugate of the W183C mutant of perfoms well as areagentless, fluorescently responsive sensor. This endosteric mutantreplaces the tryptophan residue at position 183, which makes extensivevan der Waals contacts with bound glucose or galactose. The oppositeface of the sugar pyranose ring makes similarly extensive contacts withthe phenylalanine residue at position 16. To test whether a similarendosteric mutant could function as an effective ratiometric sensor, theF16C mutant was constructed by total gene synthesis, and its acrylodanand badan conjugates were prepared. Both conjugates exhibited exhibitedexcellent ratiometric signals in response to glucose. The fluorescentlandscapes of both conjugates (FIG. 22 ) bear a remarkable resemblanceto the behavior of the GGBPI 83C-acrylodan conjugate (FIG. 5 ). Theseconjugates therefore can be used in optrodes or other sensors in thesame manner as the GGBP183C-acrylodan. The glucose affinities of theGGBP16C-acrylodan and GGBP16C-badan conjugates are ˜0.2 mM at 25° C. Itis anticipated that the affinity of these sensors also can be tuned bymutagenesis.

Example 3 Glucose and Galactose Affinities

In addition to the Roche LightCycler, which has a limited choice ofemission wavelengths, data for selected mutants also was measured atroom temperature on a Nanodrop3300 (Thermo Scientific) fluorimeter,which records full emission spectra. Emission intensities at aparticular wavelength were extracted by integration of the spectralemission intensities over a 20-nm interval centered on that wavelength.Results are shown in TABLE 6.

TABLE 6 Glucose and galactose affinities of ecGBP_183C mutants labeledwith Acrylodana. Emission Affinities Emission Affinities (nm) (mM)^(d)wavelength (mM)^(d) Response wavelength Glucose (nm) Galactose MutationShape^(b) Intensity^(c) A₁ A₂ aPPKa trueKa Ai A2 ^(app)K_(d)^(true)K_(d) — d — 483 507 6.0 4.4 483 510 31 24 E149K d — 483 507 1.51.2 483 510 34 27 E149Q d — 483 491 0.6 0.43 483 510 2.3 2.1 E149S d —483 519 0.38 0.36 487 515 6.8^(e) 5.4^(e) H152F d — 483 511 15 16 483510 nb nb H152N d — 487 495 20 15 483 510 21^(e) 21^(e) H152Q d — 487511 4.6 5.1 487 515 nb nb A155F m — 511 466 0.06 0.06 491 511 0.82 0.93A155M m — 507 466 0.05 0.05 491 520 6.7^(e) 8.7^(e) A155N d — 487 5100.14 0.23 491 515 4.5 6.6 A155S m? — 495 511 0.31 0.35 495 510 nb nbN211Q d — 479 510 29 22 479 510 nb nb ^(a)Signals S. ^(b)m,monochromatic; d, dichromatic (i.e. spectral shape changes); 0, nochange in the glucose titration. ^(c)+, increases in response to ligand;decreases; 0, no change in the glucose titration. ^(d)Blank entries, nomeasurements; nb, no binding. ^(e)Noisy data.

Signals S (either single-wavelength emission intensities, LT, or ratiosof intensities at two wavelengths, Rig) were fit to ligand-bindingisotherms:

S= ^(apo)β(1− y )+^(sat) βy   29

where ^(apo)β and ^(sat)β are the baselines in the ligand-free andligand-bound states, respectively, and y is the fractional occupancy ofthe binding sites. Baseline functions can be constant (^(apo)β) orlinearly dependent on ligand (^(sat)β). For a single ligand-bindingsite, the fractional saturation is given by

$\begin{matrix}{\overset{\_}{y} = \frac{\lbrack L\rbrack}{\lbrack L\rbrack + K_{d}}} & 30\end{matrix}$

where [L] is the ligand concentration and K_(d) the dissociationconstant corresponding to ^(app)K_(d) or ^(true)K_(d) for fits to R₁₂(weighted by the relative contributions of ^(sat)β at the two differentwavelengths) and I_(λ) respectively. For a given isothermal titration,values for ^(app)K_(d) and ^(true) K_(d) were obtained using anon-linear fitting algorithm in which these two parameters weresimultaneously fit to the three experimental binding isotherms usingequations 29 and 30, with the two monochromatic isotherms sharing thesame ^(true)K_(d) value. Three separate pairs of ^(apo)β and ^(sat)βwere fit in this procedure.

A ratiometric signal at a given point in a titration series, R₁₂(t), isgiven by the ratio of intensities at two wavelengths, ^(obs)I(λ₁,t),^(obs)I(λ₂,t) in the emission spectrum measured at that point:

$\begin{matrix}{{R_{12}(t)} = \frac{a_{t}^{obs}{I\left( {\lambda_{1},t} \right)}}{a_{t}^{obs}{I\left( {\lambda_{2},t} \right)}}} & 31\end{matrix}$

where a_(t) is an attenuation factor that describes the effect ofvariations in sample size (i.e. the amount of observable fluorophore) inthe t^(th) sample on the wavelength-independent intensity of the entireemission spectrum. Following a fit of the titration series usingequations 29 and 30, at values can be recovered by taking the averagecomparison of the observed and calculated intensities at the twowavelengths:

$\begin{matrix}{a_{t} = {\frac{1}{2}\left( {\frac{{\,^{calc}I}\left( {\lambda_{1},t} \right)}{{\,^{obs}I}\left( {\lambda_{1},t} \right)} + \frac{{\,^{calc}I}\left( {\lambda_{2},t} \right)}{{\,^{obs}I}\left( {\lambda_{2},t} \right)}} \right)}} & 32\end{matrix}$

The a_(t) value can then be applied to all wavelengths to obtain anemission spectrum of the t^(th) titration point corrected for variationsin sample size:

^(corr) I(λ)=a _(t) ^(obs) I(λ)  33

where ^(corr)I(λ) and ^(obs)I(λ) are the wavelength-dependentintensities of the corrected and observed emission spectra,respectively.

Note that the ^(app)K_(d) values are dependent on the choice of theratiometric wavelengths:

$\begin{matrix}{{\,^{app}K_{d}} = {{\,^{true}K_{d}}\frac{\,^{apo}I_{\lambda 2}}{\,^{sat}I_{\lambda l2}}}} & 34\end{matrix}$

where ^(apo)I_(λ2) and ^(apo)I_(λ2) are the emission intensities of themonochromatic signal at I₂ of the ligand-free and ligand-bound protein,respectively.

The foregoing description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent disclosure. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary aspects, but should be defined onlyin accordance with the following claims and their equivalents.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A biosensor comprising a) a polypeptide comprising a ligandbinding site and (i) one or more mutations as compared to SEQ ID NO:112(wild-type E. coli GGBP) that alter the ligand binding affinity of thepolypeptide; and b) a reporter conjugated to the polypeptide, whereinwhen the polypeptide consists of a single mutation, the single mutationis F16C, wherein the ligand-bound biosensor results in areporter-generated signal that is different from the unbound biosensor,and wherein the ligand is selected from the group consisting of glucose,galactose, and a combination thereof.

Clause 2. The biosensor of clause 1, wherein the reporter is conjugatedto F16C.

Clause 3. The biosensor of clause 1, wherein the polypeptide furthercomprises (ii) at least one additional mutation that replaces an aminoacid with a cysteine.

Clause 4. The biosensor of clause 3, wherein the reporter is conjugatedto the cysteine.

Clause 5. The biosensor of any one of the preceding clauses, wherein thebiosensor comprises a single reporter.

Clause 6. The biosensor of any one of the preceding clauses, wherein thereporter comprises a fluorophore and wherein the signal is a fluorescentsignal.

Clause 7. The biosensor of clause 6, wherein the fluorophore is selectedfrom the group consisting of acrylodan and badan.

Clause 8. The biosensor of clause 6, wherein the signal comprises anemission intensity of the fluorophore recorded at one or morewavelengths.

Clause 9. The biosensor of clause 6, wherein the change in signalcomprises a shift in the one or more wavelengths.

Clause 10. The biosensor of clause 6, wherein the signal comprises aratio of emission intensities recorded at two or more wavelengths.

Clause 11. The biosensor of clause 6, wherein the change in signalcomprises a shift in two or more wavelengths.

Clause 12. The biosensor of any one of clauses 3-11, wherein the atleast one additional mutation (ii) is W183C.

Clause 13. The biosensor of clause 12, wherein the reporter isconjugated to W183C.

Clause 14. The biosensor of any one of clauses 3-13, wherein eachmutation (i) is a mutation to an amino acid selected from the groupconsisting of Y10, D14, F16, N91, K92, E149, H152, D154, A155, R158,M182, N211, D236, and N256, and combinations thereof.

Clause 15. The biosensor of any one of clauses 3-14, wherein eachmutation (i) is selected from the group consisting of Y10A, D14A, D14Q,D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, F16L, F16A, N91A, K92A,E149K, E149Q, E149S, H152A, H152F, H152Q, H152N, D154A, D154N, A155S,A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, R158A,R158K, M182W, N211F, N211W, N211K, N211Q, N211S, N211H, N211M, D236A,D236N, N256A, and N256D, and combinations thereof.

Clause 16. The biosensor of any one of clauses 3-15, wherein themutation (i) affects the interaction of the polypeptide with boundglucose, wherein the interaction is with a portion of the glucoseselected from the group consisting of 1-hydroxyl, 2-hydroxyl,3-hydroxyl, 4-hydoxyl, 6-hydroxyl, pyranose ring, and combinationsthereof.

Clause 17. The biosensor of any one of clauses 3-16, wherein themutation (i) affects the interaction of the mutant polypeptide with thereporter group.

Clause 18. The biosensor of any one of clauses 3-17, wherein themutation (i) affects the interaction of the mutant polypeptide with awater molecule.

Clause 19. The biosensor of any one of the preceding clauses, whereinthe polypeptide has an affinity (K_(D)) for glucose within theconcentration range of glucose in vivo for a subject.

Clause 20. The biosensor of any one of the preceding clauses, whereinthe polypeptide has an affinity (K_(D)) for galactose within theconcentration range of galactose in vivo for a subject.

Clause 21. The biosensor of any one of clauses 19 and 20, wherein thesubject is a mammal.

Clause 22. The biosensor of any one of clauses 19 and 20, wherein thesubject is a primate or non-primate.

Clause 23. The biosensor of clause 22, wherein the subject is anon-primate selected from a cow, pig, camel, llama, horse, goat, rabbit,sheep, hamsters, guinea pig, cat, dog, rat, and mouse.

Clause 24. The biosensor of clause 22, wherein the subject is a primateselected from a monkey, chimpanzee, and human.

Clause 25. The biosensor of clause 24, wherein the subject is a human.

Clause 26. The biosensor of any one of the preceding clauses, whereinthe polypeptide has an affinity (K_(D)) for glucose in the range ofabout 0.2 mM to about 100 mM.

Clause 27. The biosensor of any one of the preceding clauses, whereinthe polypeptide has an affinity (K_(D)) for galactose in the range ofabout 0.8 mM to about 100 mM.

Clause 28. The biosensor of any one of the preceding clauses, whereinthe biosensor is capable of detecting glucose in the hypoglycemic,hyperglycemic, and hyperglycemic-hyperosmotic ranges.

Clause 29. The biosensor of any one of the preceding clauses, whereinthe biosensor is capable of detecting glucose in the range of about 0.1mmol/L to about 120 mmol/L.

Clause 30. The biosensor of any one of the preceding clauses, whereinthe biosensor is capable of detecting glucose in the range of about 4mmol/L to about 33 mmol/L.

Clause 31. The biosensor of any one of the preceding clauses, whereinthe biosensor is capable of detecting galactose in the range of about0.2 mM to about 400 mM.

Clause 32. The biosensor of any one of the preceding clauses, whereinthe mutant polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-54.

Clause 33. A polypeptide comprising the amino acid sequence of SEQ IDNO: 1.

Clause 34. A polypeptide comprising the amino acid sequence of SEQ IDNO: 2.

Clause 35. A polypeptide comprising the amino acid sequence of SEQ IDNO: 3.

Clause 36. A polypeptide comprising the amino acid sequence of SEQ IDNO: 4.

Clause 37. A polypeptide comprising the amino acid sequence of SEQ IDNO: 5.

Clause 38. A polypeptide comprising the amino acid sequence of SEQ IDNO: 6.

Clause 39. A polypeptide comprising the amino acid sequence of SEQ IDNO: 7.

Clause 40. A polypeptide comprising the amino acid sequence of SEQ IDNO: 8.

Clause 41. A polypeptide comprising the amino acid sequence of SEQ IDNO: 9.

Clause 42. A polypeptide comprising the amino acid sequence of SEQ IDNO: 10.

Clause 43. A polypeptide comprising the amino acid sequence of SEQ IDNO: 11.

Clause 44. A polypeptide comprising the amino acid sequence of SEQ IDNO: 12.

Clause 45. A polypeptide comprising the amino acid sequence of SEQ IDNO: 13.

Clause 46. A polypeptide comprising the amino acid sequence of SEQ IDNO: 14.

Clause 47. A polypeptide comprising the amino acid sequence of SEQ IDNO: 15.

Clause 48. A polypeptide comprising the amino acid sequence of SEQ IDNO: 16.

Clause 49. A polypeptide comprising the amino acid sequence of SEQ IDNO: 17.

Clause 50. A polypeptide comprising the amino acid sequence of SEQ IDNO: 18.

Clause 51. A polypeptide comprising the amino acid sequence of SEQ IDNO: 19.

Clause 52. A polypeptide comprising the amino acid sequence of SEQ IDNO: 20.

Clause 53. A polypeptide comprising the amino acid sequence of SEQ IDNO: 21.

Clause 54. A polypeptide comprising the amino acid sequence of SEQ IDNO: 22.

Clause 55. A polypeptide comprising the amino acid sequence of SEQ IDNO: 23.

Clause 56. A polypeptide comprising the amino acid sequence of SEQ IDNO: 24.

Clause 57. A polypeptide comprising the amino acid sequence of SEQ IDNO: 25.

Clause 58. A polypeptide comprising the amino acid sequence of SEQ IDNO: 26.

Clause 60. A polypeptide comprising the amino acid sequence of SEQ IDNO: 27.

Clause 61. A polypeptide comprising the amino acid sequence of SEQ IDNO: 28.

Clause 62. A polypeptide comprising the amino acid sequence of SEQ IDNO: 29.

Clause 63. A polypeptide comprising the amino acid sequence of SEQ IDNO: 30.

Clause 64. A polypeptide comprising the amino acid sequence of SEQ IDNO: 31.

Clause 65. A polypeptide comprising the amino acid sequence of SEQ IDNO: 32.

Clause 66. A polypeptide comprising the amino acid sequence of SEQ IDNO: 33.

Clause 67. A polypeptide comprising the amino acid sequence of SEQ IDNO: 34.

Clause 68. A polypeptide comprising the amino acid sequence of SEQ IDNO: 35.

Clause 69. A polypeptide comprising the amino acid sequence of SEQ IDNO: 36.

Clause 70. A polypeptide comprising the amino acid sequence of SEQ IDNO: 37.

Clause 71. A polypeptide comprising the amino acid sequence of SEQ IDNO: 38.

Clause 72. A polypeptide comprising the amino acid sequence of SEQ IDNO: 39.

Clause 73. A polypeptide comprising the amino acid sequence of SEQ IDNO: 40.

Clause 74. A polypeptide comprising the amino acid sequence of SEQ IDNO: 41.

Clause 75. A polypeptide comprising the amino acid sequence of SEQ IDNO: 42.

Clause 76. A polypeptide comprising the amino acid sequence of SEQ IDNO: 43.

Clause 77. A polypeptide comprising the amino acid sequence of SEQ IDNO: 44.

Clause 78. A polypeptide comprising the amino acid sequence of SEQ IDNO: 45.

Clause 79. A polypeptide comprising the amino acid sequence of SEQ IDNO: 46.

Clause 80. A polypeptide comprising the amino acid sequence of SEQ IDNO: 47.

Clause 81. A polypeptide comprising the amino acid sequence of SEQ IDNO: 48.

Clause 82. A polypeptide comprising the amino acid sequence of SEQ IDNO: 49.

Clause 83. A polypeptide comprising the amino acid sequence of SEQ IDNO: 50.

Clause 84. A polypeptide comprising the amino acid sequence of SEQ IDNO: 51.

Clause 85. A polypeptide comprising the amino acid sequence of SEQ IDNO: 52.

Clause 86. A polypeptide comprising the amino acid sequence of SEQ IDNO: 53.

Clause 87. A polypeptide comprising the amino acid sequence of SEQ IDNO: 54.

Clause 88. A polynucleotide encoding the polypeptide of any one of thepreceding clauses.

Clause 89. The polynucleotide of clause 88, wherein the polynucleotidecomprises at least one sequence selected from the group consisting ofSEQ ID NOs: 56-109.

Clause 90. A vector comprising the polynucleotide of clause 88 or 89.

Clause 91. A panel comprising a plurality of biosensors according to anyone of clauses 1-32.

Clause 92. The panel of clause 91, wherein the panel comprises acomposite sensor or an array.

Clause 93. The panel of clause 92, wherein the array is selected from amultichannel array or multiplexed array.

Clause 94. The panel of any one of clauses 91-93, wherein each biosensorcomprises the same reporter group.

Clause 95. The panel of any one of clauses 91-93, wherein each biosensorcomprises a different reporter group.

Clause 96. The panel of clause 92, wherein the array comprises aplurality of sensor elements, each sensor element comprising a biosensordifferent from or the same as those of the other sensor elements.

Clause 97. The panel of clause 92, wherein the composite sensorcomprises a plurality of sensor elements, each sensor element comprisinga mixture of different biosensors.

Clause 98. The panel of clause 92, wherein the composite sensorcomprises a single sensor element, the single sensor element comprisinga mixture of different biosensors.

Clause 99. A method of determining the concentration of glucose,galactose, or a combination thereof, in a sample from a subject, themethod comprising applying the sample to the panel of any one of clauses91-97.

Clause 100. The method of clause 99, wherein the sample is from asubject.

Clause 101. The method according to clause 99 or 100, wherein the samplecomprises a biological fluid.

Clause 102. The method according to clause 101, wherein the biologicalfluid is selected from the group consisting of blood, urine,interstitial fluid, saliva, sweat, tears, gastric lavage, fecal matter,emesis, bile, or combinations thereof.

Clause 103. The method according to clause 100, wherein the samplecomprises skin.

Clause 104. A method of detecting the presence of a ligand in a sample,the method comprising a) contacting the biosensor of any one of clauses1-32 with the sample; b) measuring a signal from the biosensor; and c)comparing the signal to a ligand-free control, wherein a difference insignal indicates the presence of ligand in the sample, and wherein theligand is selected from the group consisting of glucose, galactose, anda combination thereof.

Clause 105. A method of determining the concentration of a ligand in asample, the method comprising a) contacting the biosensor of any one ofclauses 1-32 with the sample; b) measuring a signal from the biosensor;and c) comparing the signal to a standard hyperbolic ligand bindingcurve to determine the concentration of ligand in the test sample,wherein the standard hyperbolic ligand binding curve is prepared bymeasuring the signal transduced by the biosensor when contacted withcontrol samples containing known concentrations of ligand, and whereinthe ligand is selected from the group consisting of glucose, galactose,and a combination thereof.

Clause 106. A method of episodically or continuously monitoring thepresence of a ligand in a reaction, the method comprising a) contactingthe biosensor of any one of clauses 1-32 with the reaction; b)maintaining the reaction under conditions such that the polypeptide iscapable of binding ligand present in the reaction; and c) episodicallyor continuously monitoring the signal from the biosensor in thereaction, wherein the ligand is selected from the group consisting ofglucose, galactose, and a combination thereof.

Clause 107. A method of episodically or continuously monitoring thepresence of a ligand in a reaction, the method comprising a) contactingthe biosensor of any one of clauses 1-32 with the reaction; b)maintaining the reaction under conditions such that the polypeptide iscapable of binding ligand present in the reaction; c) episodically orcontinuously monitoring the signal from the biosensor in the reaction;and d) comparing the signal to a standard hyperbolic ligand bindingcurve to determine the concentration of ligand in the test sample,wherein the standard hyperbolic ligand binding curve is prepared bymeasuring the signal transduced by the biosensor when contacted withcontrol samples containing known concentrations of ligand, wherein theligand is selected from the group consisting of glucose, galactose, anda combination thereof.

Clause 108. The method of any one of clauses 104-107, wherein thebiosensor is placed in contact with a subject's skin or mucosal surface.

Clause 109. The method of any one of clauses 104-107, wherein thebiosensor is implanted in a subject's body.

Clause 110. The method of any one of clauses 104-107, wherein thebiosensor is implanted in a subject's blood vessel, vein, eye, naturalor artificial pancreas, alimentary canal, stomach, intestine, esophagus,or skin.

Clause 111. The method of any one of clauses 104-107, wherein thebiosensor is configured within or on the surface of a contact lens.

Clause 112. The method of any one of clauses 104-107, wherein thebiosensor is configured to be implanted in the skin.

Clause 113. The method of any one of clauses 104-107, wherein thebiosensor is implanted in a subject with an optode.

Clause 114. The method of any one of clauses 104-107, wherein thebiosensor is implanted in a subject with a microbead.

Clause 115. The method of any one of clauses 104-107, wherein thebiosensor generates the signal transdermally.

Clause 116. The method of clause 106, wherein the method furthercomprises d) comparing the signal to a ligand-free control, wherein adifference in signal indicates the presence of ligand in the reaction.

Clause 117. The method of clause 106, wherein the method furthercomprises d) comparing the signal to a standard hyperbolic ligandbinding curve to determine the concentration of ligand in the testsample, wherein the standard hyperbolic ligand binding curve is preparedby measuring the signal transduced by the biosensor when contacted withcontrol samples containing known concentrations of ligand.

Clause 118. The method of any one of clauses 99, 104-107, and 116-117,wherein the sample comprises a fermentation sample.

Clause 119. The method of any one of clauses 99, 104-107, and 116-117,wherein the sample comprises food or beverage.

Clause 120. The method of clause 119, wherein the sample comprises abeverage selected from soft drink, fountain beverage, water, coffee,tea, milk, dairy-based beverage, soy-based beverage, almond-basedbeverage, vegetable juice, fruit juice, fruit juice flavored drink,energy drink, sport drink, and alcoholic product, and combinationsthereof.

Clause 121. The method of clause 120, wherein the sample comprises waterselected from flavored water, mineral water, spring water, sparklingwater, and tonic water, and combinations thereof.

Clause 122. The method of clause 120, wherein the sample comprises analcoholic product selected from beer, malt beverage, liqueur, whiskey,and wine, and combinations thereof.

Clause 123. The method of clause 119, wherein the sample comprises foodcomprising a semi-solid or liquid form.

Clause 124. The method of clause 119 wherein the sample comprises foodselected from yogurt, soup, ice cream, broth, purees, shakes, smoothies,batter, condiments, and sauce, and combinations thereof.

Clause 125. The method of any one of clauses 99, 103-106, and 115-122,wherein the sample is from food engineering.

SEQUENCES GGBP183C polypeptide SEQ ID NO: 1MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF GGBP16C polypeptide SEQ ID NO: 2MADTRIGVTIYKYDDNCMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF GGBP183C14A polypeptide SEQ ID NO: 3MADTRIGVTIYKYDANFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C152N polypeptide SEQ ID NO: 4MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGNPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWICTDNKVVRVPYVGVDKDNLAEFSKK GGBP183C152F polypeptide SEQ ID NO: 5MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGFPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C152Q polypeptide SEQ ID NO: 6MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGQPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C149Q polypeptide SEQ ID NO: 7MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVLIKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRIZALDS YDKAYYVGTDSKESGIIQGDAKHWAANQGWDLNKDGQIQFVLLKGQPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALA LVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C149S polypeptide SEQ ID NO: 8MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGSPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C149K polypeptide SEQ ID NO: 9MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGKPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C155N polypeptide SEQ ID NO: 10MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDNEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK GGBP183C155H polypeptide SEQ ID NO: 11MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDHEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK A155F + 183C polypeptide SEQ ID NO: 12MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDFEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155K + 183C polypeptide SEQ ID NO: 13MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDKEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155L + 183C polypeptide SEQ ID NO: 14MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDLEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155M + 183C polypeptide SEQ ID NO: 15MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDMEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155Q + 183C polypeptide SEQ ID NO: 16MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDQEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155S + 183C polypeptide SEQ ID NO: 17MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDSEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155W + 183C polypeptide SEQ ID NO: 18MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDWEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF A155Y + 183C polypeptide SEQ ID NO: 19MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDYEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14E + 183C polypeptide SEQ ID NO: 20MADTRIGVTIYKYDENFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14F + 183C polypeptide SEQ ID NO: 21MADTRIGVTIYKYDFNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKJEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14H + 183C polypeptide SEQ ID NO: 22MADTRIGVTIYKYDHNFMSVVRIZAIEQDAKAAPD VQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEIZARGQNVPVVFFNKEPSRKALDSY DIZAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGI KTEQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEA LALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWIUDNKVVRVPYVGVDKDNLAEF D14L + 183C polypeptide SEQ ID NO: 23MADTRIGVTIYKYDLNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14N + 183C polypeptide SEQ ID NO: 24MADTRIGVTIYKYDNNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14Q + 183C polypeptide SEQ ID NO: 25MADTRIGVTIYKYDQNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14S + 183C polypeptide SEQ ID NO: 26MADTRIGVTIYKYDSNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14T + 183C polypeptide SEQ ID NO: 27MADTRIGVTIYKYDTNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D14Y + 183C polypeptide SEQ ID NO: 28MADTRIGVTIYKYDYNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D154A + 183C polypeptide SEQ ID NO: 29MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPAAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D154N + 183C polypeptide SEQ ID NO: 30MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPNAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWIGDNKVVRVPYVGVDKDNLAEF D236A + 183C polypeptide SEQ ID NO: 31MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANIGEVVIANNDAMAMGAVEALKAHNKSSIPVFGVAALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF D236N + 183C polypeptide SEQ ID NO: 32MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVNALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF E149Q + 183C polypeptide SEQ ID NO: 33MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGQPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF F16A + 183C polypeptide SEQ ID NO: 34MADTRIGVTIYKYDDNAMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF F16L + 183C polypeptide SEQ ID NO: 35MADTRIGVTIYKYDDNLMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF H152A + 183C polypeptide SEQ ID NO: 36MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGAPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF H152K + 183C polypeptide SEQ ID NO: 37MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGKPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF K92A + 183C polypeptide SEQ ID NO: 38MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNAEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF M182W + 183C polypeptide SEQ ID NO: 39MADTRIGVTIYKYDDNFMSVVRIZAIEQDAKAAPD VQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYD KAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAWCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALA LVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211A + 183C polypeptide SEQ ID NO: 40MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANADAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAIZATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211F + 183C polypeptide SEQ ID NO: 41MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANFDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211H + 1830 polypeptide SEQ ID NO: 42MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANHDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211K + 1830 polypeptide SEQ ID NO: 43MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANKDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211M + 1830 polypeptide SEQ ID NO: 44MADTRIGVTIYKYDDNFMSVVRKAIEQDAIZAAPD VQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYD KAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANMDAMAMGAVEALKAHNKSSIPVFGVDALPEALA LVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211Q + 183C polypeptide SEQ ID NO: 45MADTRIGVTIYKYDDNFMSVVRIZAIEQDAKAAPD VQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYD KAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANQDAMAMGAVEALKAHNKSSIPVFGVDALPEALA LVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211S + 1830 polypeptide SEQ ID NO: 46MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANSDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211W + 183C polypeptide SEQ ID NO: 47MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRIZALDSYD IZAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIK TEQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANWDAMAMGAVEALKAHNKSSIPVFGVDALPEAL ALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N211Y + 183C polypeptide SEQ ID NO: 48MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVIZALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYD KAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANYDAMAMGAVEALIZAHNKSSIPVFGVDALPEAL ALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N256A + 183C polypeptide SEQ ID NO: 49MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRIZALDSYD KAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALA LVKSGALAGTVLADANNQAKATFDLAKNLADGKGAADGTNWIGDNKVVRVPYVGVDKDNLAEF N256D + 183C polypeptide SEQ ID NO: 50MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLDDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF N91A + 183C polypeptide SEQ ID NO: 51MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFAKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF R158A + 183C polypeptide SEQ ID NO: 52MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEAATTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF R158K + 183C polypeptide SEQ ID NO: 53MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEAKTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWIGDNKVVRVPYVGVDKDNLAEF Y10A + 183C polypeptide SEQ ID NO: 54MADTRIGVTIAKYDDNFMSVVRKAIEQDAKAAPDV QLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDK AYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTE QLQLDTAMCDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALAL VKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEF Wild-type E. coli GGBP polypeptideSEQ ID NO: 55 MADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVD PAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDG QIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIA NNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAA DGTNWKIDNKVVRVPYVGVDKDNLAEF183C polynucleotide SEQ ID NO: 56ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGTGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT16C polynucleotide SEQ ID NO: 57ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTGTATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGGGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14A polynucleotide SEQ ID NO: 58ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGCGAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT152N polynucleotide SEQ ID NO: 59ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTAACCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT152F polynucleotide SEQ ID NO: 60ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTTTTCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT1520 polynucleotide SEQ ID NO: 61ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCAGCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT1490 polynucleotide SEQ ID NO: 62ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCCAGCCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT149S polynucleotide SEQ ID NO: 63ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCAGCCCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT149K polynucleotide SEQ ID NO: 64ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCAAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155N polynucleotide SEQ ID NO: 65ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATAACGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155H polynucleotide SEQ ID NO: 66ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATCATGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155F polynucleotide SEQ ID NO: 67ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATTTTGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155K polynucleotide SEQ ID NO: 68ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATAAAGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155L polynucleotide SEQ ID NO: 69ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATCTGGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155M polynucleotide SEQ ID NO: 70ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATATGGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155Q polynucleotide SEQ ID NO: 71ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATCAGGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155S polynucleotide SEQ ID NO: 72ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATAGCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155W polynucleotide SEQ ID NO: 73ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATTGGGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT155Y polynucleotide SEQ ID NO: 74ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATTATGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14E polynucleotide SEQ ID NO: 75ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGAAAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14F polynucleotide SEQ ID NO: 76ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATTTTAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14H polynucleotide SEQ ID NO: 77ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATCATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14L polynucleotide SEQ ID NO: 78ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATCTGAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14N polynucleotide SEQ ID NO: 79ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATAACAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14Q polynucleotide SEQ ID NO: 80ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATCAGAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14S polynucleotide SEQ ID NO: 81ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATAGCAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14T polynucleotide SEQ ID NO: 82ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATACCAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT14Y polynucleotide SEQ ID NO: 83ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATTATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT154A polynucleotide SEQ ID NO: 84ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGCGGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT154N polynucleotide SEQ ID NO: 85ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGAACGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT236A polynucleotide SEQ ID NO: 86ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGCGGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT236N polynucleotide SEQ ID NO: 87ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGAACGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT149Q polynucleotide SEQ ID NO: 88ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCCAGCCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT16A polynucleotide SEQ ID NO: 89ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATGCGATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT16L polynucleotide SEQ ID NO: 90ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATCTGATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT152A polynucleotide SEQ ID NO: 91ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTGCGCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT152K polynucleotide SEQ ID NO: 92ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTAAACCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT92A polynucleotide SEQ ID NO: 93ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACGCGGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT182W polynucleotide SEQ ID NO: 94ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGTGGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211A polynucleotide SEQ ID NO: 95ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACGCGGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211F polynucleotide SEQ ID NO: 96ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACTTTGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211H polynucleotide SEQ ID NO: 97ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACCATGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211K polynucleotide SEQ ID NO: 98ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAAAGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211M polynucleotide SEQ ID NO: 99ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACATGGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT2110 polynucleotide SEQ ID NO: 100ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACCAGGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211S polynucleotide SEQ ID NO: 101ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAGCGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211W polynucleotide SEQ ID NO: 102ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACTGGGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT211Y polynucleotide SEQ ID NO: 103ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACTATGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT256A polynucleotide SEQ ID NO: 104ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGGCGGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT256D polynucleotide SEQ ID NO: 105ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGGATGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT92A polynucleotide SEQ ID NO: 106ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACGCGGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT158A polynucleotide SEQ ID NO: 107ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGGCGACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT158K polynucleotide SEQ ID NO: 108ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGAAAACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTT10A polynucleotide SEQ ID NO: 109ATGGCAGATACTCGTATTGGTGTAACTATTGCGAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGCGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTTWild-type E. coli GGBP polynucleotide SEQ ID NO: 110ATGGCAGATACTCGTATTGGTGTAACTATTTATAAATACGATGATAATTTCATGAGCGTAGTACGTAAAGCAATTGAACAAGATGCGAAAGCGGCCCCGGATGTTCAGCTGCTGATGAACGATAGCCAGAACGATCAGAGCAAACAGAACGATCAGATTGATGTGCTGCTGGCCAAAGGCGTGAAAGCCCTGGCCATTAACCTGGTTGATCCGGCGGCGGCCGGTACCGTTATTGAAAAAGCCCGTGGCCAGAACGTGCCGGTGGTGTTCTTCAACAAAGAACCGAGCCGCAAAGCGCTGGATAGCTACGATAAAGCGTACTATGTGGGCACCGATAGCAAAGAAAGCGGCATTATTCAGGGCGATCTGATTGCGAAACATTGGGCGGCGAACCAGGGCTGGGATCTGAACAAAGATGGCCAGATTCAGTTCGTGCTGCTGAAAGGCGAACCGGGTCATCCGGATGCCGAAGCGCGTACCACCTATGTGATCAAAGAACTGAACGACAAAGGCATCAAAACCGAACAGCTGCAACTGGATACCGCGATGTGGGATACCGCGCAGGCGAAAGATAAAATGGATGCGTGGCTGAGCGGTCCGAACGCGAACAAAATTGAAGTGGTGATTGCGAACAACGATGCGATGGCGATGGGCGCGGTGGAAGCGCTGAAAGCCCATAACAAATCCAGCATTCCGGTGTTTGGCGTGGATGCCCTGCCGGAAGCGCTGGCGCTGGTTAAAAGCGGTGCGCTGGCGGGCACCGTTCTGAACGATGCCAACAACCAGGCGAAAGCCACCTTCGATCTGGCGAAAAACCTGGCGGATGGTAAAGGCGCGGCCGATGGCACCAACTGGAAAATTGATAACAAAGTGGTGCGTGTGCCGTATGTGGGCGTGGAT AAAGATAACCTGGCCGAATTTHistine tag, polypeptide SEQ ID NO: 111 GGSHHHHHHWild-type E. coli GGBP polypeptide SEQ ID NO: 112ADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDN KVVRVPYVGVDKDNLAEF

We claim:
 1. A biosensor comprising: a) a polypeptide comprising aligand binding site and (i) one or more mutations as compared to SEQ IDNO:112 (wild-type E. coli GGBP) that alter the ligand binding affinityof the polypeptide; and b) a reporter conjugated to the polypeptide,wherein when the polypeptide consists of a single mutation, the singlemutation is F16C, wherein the ligand-bound biosensor results in areporter-generated signal that is different from the unbound biosensor,and wherein the ligand is selected from the group consisting of glucose,galactose, and a combination thereof.
 2. The biosensor of claim 1,wherein the reporter is conjugated to F16C.
 3. The biosensor of claim 1,wherein the polypeptide further comprises (ii) at least one additionalmutation that replaces an amino acid with a cysteine.
 4. The biosensorof claim 3, wherein the reporter is conjugated to the cysteine.
 5. Thebiosensor of any one of the preceding claims, wherein the biosensorcomprises a single reporter.
 6. The biosensor of any one of thepreceding claims, wherein the reporter comprises a fluorophore andwherein the signal is a fluorescent signal.
 7. The biosensor of claim 6,wherein the fluorophore is selected from the group consisting ofacrylodan and badan.
 8. The biosensor of claim 6, wherein the signalcomprises an emission intensity of the fluorophore recorded at one ormore wavelengths.
 9. The biosensor of claim 6, wherein the change insignal comprises a shift in the one or more wavelengths.
 10. Thebiosensor of claim 6, wherein the signal comprises a ratio of emissionintensities recorded at two or more wavelengths.
 11. The biosensor ofclaim 6, wherein the change in signal comprises a shift in two or morewavelengths.
 12. The biosensor of any one of claims 3-11, wherein the atleast one additional mutation (ii) is W183C.
 13. The biosensor of claim12, wherein the reporter is conjugated to W183C.
 14. The biosensor ofany one of claims 3-13, wherein each mutation (i) is a mutation to anamino acid selected from the group consisting of Y10, D14, F16, N91,K92, E149, H152, D154, A155, R158, M182, N211, D236, and N256, andcombinations thereof.
 15. The biosensor of any one of claims 3-14,wherein each mutation (i) is selected from the group consisting of Y10A,D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, F16L, F16A,N91A, K92A, E149K, E149Q, E149S, H152A, H152F, H152Q, H152N, D154A,D154N, A155S, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W,A155Q, R158A, R158K, M182W, N211F, N211W, N211K, N211Q, N211S, N211H,N211M, D236A, D236N, N256A, and N256D, and combinations thereof.
 16. Thebiosensor of any one of claims 3-15, wherein the mutation (i) affectsthe interaction of the polypeptide with bound glucose, wherein theinteraction is with a portion of the glucose selected from the groupconsisting of 1-hydroxyl, 2-hydroxyl, 3-hydroxyl, 4-hydroxyl,6-hydroxyl, pyranose ring, and combinations thereof.
 17. The biosensorof any one of claims 3-16, wherein the mutation (i) affects theinteraction of the mutant polypeptide with the reporter group.
 18. Thebiosensor of any one of claims 3-17, wherein the mutation (i) affectsthe interaction of the mutant polypeptide with a water molecule.
 19. Thebiosensor of any one of the preceding claims, wherein the polypeptidehas an affinity (K_(D)) for glucose within the concentration range ofglucose in vivo for a subject.
 20. The biosensor of any one of thepreceding claims, wherein the polypeptide has an affinity (K_(D)) forgalactose within the concentration range of galactose in vivo for asubject.
 21. The biosensor of any one of claims 19 and 20, wherein thesubject is a mammal.
 22. The biosensor of any one of claims 19 and 20,wherein the subject is a primate or non-primate.
 23. The biosensor ofclaim 22, wherein the subject is a non-primate selected from a cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse.
 24. The biosensor of claim 22, wherein the subjectis a primate selected from a monkey, chimpanzee, and human.
 25. Thebiosensor of claim 24, wherein the subject is a human.
 26. The biosensorof any one of the preceding claims, wherein the polypeptide has anaffinity (K_(D)) for glucose in the range of about 0.2 mM to about 100mM.
 27. The biosensor of any one of the preceding claims, wherein thepolypeptide has an affinity (K_(D)) for galactose in the range of about0.8 mM to about 100 mM.
 28. The biosensor of any one of the precedingclaims, wherein the biosensor is capable of detecting glucose in thehypoglycemic, hyperglycemic, and hyperglycemic-hyperosmotic ranges. 29.The biosensor of any one of the preceding claims, wherein the biosensoris capable of detecting glucose in the range of about 0.1 mmol/L toabout 120 mmol/L.
 30. The biosensor of any one of the preceding claims,wherein the biosensor is capable of detecting glucose in the range ofabout 4 mmol/L to about 33 mmol/L.
 31. The biosensor of any one of thepreceding claims, wherein the biosensor is capable of detectinggalactose in the range of about 0.2 mM to about 400 mM.
 32. Thebiosensor of any one of the preceding claims, wherein the mutantpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-54.
 33. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 1. 34. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 2. 35. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 3. 36. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 4. 37. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 5. 38. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 6. 39. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 7. 40. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 8. 41. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 9. 42. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 10. 43. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 11. 44. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 12. 45. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 13. 46. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 14. 47. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 15. 48. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 16. 49. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 17. 50. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 18. 51. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 19. 52. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 20. 53. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 21. 54. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 22. 55. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 23. 56. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 24. 57. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 25. 58. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 26. 60. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 27. 61. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 28. 62. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 29. 63. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 30. 64. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 31. 65. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 32. 66. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 33. 67. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 34. 68. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 35. 69. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 36. 70. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 37. 71. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 38. 72. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 39. 73. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 40. 74. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 41. 75. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 42. 76. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 43. 77. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 44. 78. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 45. 79. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 46. 80. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 47. 81. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 48. 82. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 49. 83. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 50. 84. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 51. 85. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 52. 86. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 53. 87. A polypeptide comprising the aminoacid sequence of SEQ ID NO:
 54. 88. A polynucleotide encoding thepolypeptide of any one of the preceding claims.
 89. The polynucleotideof claim 88, wherein the polynucleotide comprises at least one sequenceselected from the group consisting of SEQ ID NOs: 56-109.
 90. A vectorcomprising the polynucleotide of claim 88 or
 89. 91. A panel comprisinga plurality of biosensors according to any one of claims 1-32.
 92. Thepanel of claim 91, wherein the panel comprises a composite sensor or anarray.
 93. The panel of claim 92, wherein the array is selected from amultichannel array or multiplexed array.
 94. The panel of any one ofclaims 91-93, wherein each biosensor comprises the same reporter group.95. The panel of any one of claims 91-93, wherein each biosensorcomprises a different reporter group.
 96. The panel of claim 92, whereinthe array comprises a plurality of sensor elements, each sensor elementcomprising a biosensor different from or the same as those of the othersensor elements.
 97. The panel of claim 92, wherein the composite sensorcomprises a plurality of sensor elements, each sensor element comprisinga mixture of different biosensors.
 98. The panel of claim 92, whereinthe composite sensor comprises a single sensor element, the singlesensor element comprising a mixture of different biosensors.
 99. Amethod of determining the concentration of glucose, galactose, or acombination thereof, in a sample from a subject, the method comprisingapplying the sample to the panel of any one of claims 91-97.
 100. Themethod of claim 99, wherein the sample is from a subject.
 101. Themethod according to claim 99 or 100, wherein the sample comprises abiological fluid.
 102. The method according to claim 101, wherein thebiological fluid is selected from the group consisting of blood, urine,interstitial fluid, saliva, sweat, tears, gastric lavage, fecal matter,emesis, bile, or combinations thereof.
 103. The method according toclaim 100, wherein the sample comprises skin.
 104. A method of detectingthe presence of a ligand in a sample, the method comprising: a)contacting the biosensor of any one of claims 1-32 with the sample; b)measuring a signal from the biosensor; and c) comparing the signal to aligand-free control, wherein a difference in signal indicates thepresence of ligand in the sample, and wherein the ligand is selectedfrom the group consisting of glucose, galactose, and a combinationthereof.
 105. A method of determining the concentration of a ligand in asample, the method comprising: a) contacting the biosensor of any one ofclaims 1-32 with the sample; b) measuring a signal from the biosensor;and c) comparing the signal to a standard hyperbolic ligand bindingcurve to determine the concentration of ligand in the test sample,wherein the standard hyperbolic ligand binding curve is prepared bymeasuring the signal transduced by the biosensor when contacted withcontrol samples containing known concentrations of ligand, and whereinthe ligand is selected from the group consisting of glucose, galactose,and a combination thereof.
 106. A method of episodically or continuouslymonitoring the presence of a ligand in a reaction, the methodcomprising: a) contacting the biosensor of any one of claims 1-32 withthe reaction; b) maintaining the reaction under conditions such that thepolypeptide is capable of binding ligand present in the reaction; and c)episodically or continuously monitoring the signal from the biosensor inthe reaction, wherein the ligand is selected from the group consistingof glucose, galactose, and a combination thereof.
 107. A method ofepisodically or continuously monitoring the presence of a ligand in areaction, the method comprising: a) contacting the biosensor of any oneof claims 1-32 with the reaction; b) maintaining the reaction underconditions such that the polypeptide is capable of binding ligandpresent in the reaction; c) episodically or continuously monitoring thesignal from the biosensor in the reaction; and d) comparing the signalto a standard hyperbolic ligand binding curve to determine theconcentration of ligand in the test sample, wherein the standardhyperbolic ligand binding curve is prepared by measuring the signaltransduced by the biosensor when contacted with control samplescontaining known concentrations of ligand, wherein the ligand isselected from the group consisting of glucose, galactose, and acombination thereof.
 108. The method of any one of claims 104-107,wherein the biosensor is placed in contact with a subject's skin ormucosal surface.
 109. The method of any one of claims 104-107, whereinthe biosensor is implanted in a subject's body.
 110. The method of anyone of claims 104-107, wherein the biosensor is implanted in a subject'sblood vessel, vein, eye, natural or artificial pancreas, alimentarycanal, stomach, intestine, esophagus, or skin.
 111. The method of anyone of claims 104-107, wherein the biosensor is configured within or onthe surface of a contact lens.
 112. The method of any one of claims104-107, wherein the biosensor is configured to be implanted in theskin.
 113. The method of any one of claims 104-107, wherein thebiosensor is implanted in a subject with an optode.
 114. The method ofany one of claims 104-107, wherein the biosensor is implanted in asubject with a microbead.
 115. The method of any one of claims 104-107,wherein the biosensor generates the signal transdermally.
 116. Themethod of claim 106, wherein the method further comprises: d) comparingthe signal to a ligand-free control, wherein a difference in signalindicates the presence of ligand in the reaction.
 117. The method ofclaim 106, wherein the method further comprises: d) comparing the signalto a standard hyperbolic ligand binding curve to determine theconcentration of ligand in the test sample, wherein the standardhyperbolic ligand binding curve is prepared by measuring the signaltransduced by the biosensor when contacted with control samplescontaining known concentrations of ligand.
 118. The method of any one ofclaims 99, 104-107, and 116-117, wherein the sample comprises afermentation sample.
 119. The method of any one of claims 99, 104-107,and 116-117, wherein the sample comprises food or beverage.
 120. Themethod of claim 119, wherein the sample comprises a beverage selectedfrom soft drink, fountain beverage, water, coffee, tea, milk,dairy-based beverage, soy-based beverage, almond-based beverage,vegetable juice, fruit juice, fruit juice flavored drink, energy drink,sport drink, and alcoholic product, and combinations thereof.
 121. Themethod of claim 120, wherein the sample comprises water selected fromflavored water, mineral water, spring water, sparkling water, and tonicwater, and combinations thereof.
 122. The method of claim 120, whereinthe sample comprises an alcoholic product selected from beer, maltbeverage, liqueur, whiskey, and wine, and combinations thereof.
 123. Themethod of claim 119, wherein the sample comprises food comprising asemi-solid or liquid form.
 124. The method of claim 119 wherein thesample comprises food selected from yogurt, soup, ice cream, broth,purees, shakes, smoothies, batter, condiments, and sauce, andcombinations thereof.
 125. The method of any one of claims 99, 103-106,and 115-122, wherein the sample is from food engineering.